Parasitic infections remain an important public health problem worldwide. Accurate identification of parasites is essential for appropriate treatment, epidemiological surveillance, and prevention of transmission. Traditional diagnostic methods, particularly microscopic examination, are still widely used; however, they may have limited sensitivity and may not reliably distinguish morphologically similar parasite species. Molecular diagnostic techniques, especially polymerase chain reaction (PCR) and real-time PCR (qPCR), have therefore become increasingly important in modern parasitology. The selection of an appropriate genetic target is one of the most important factors determining the sensitivity and specificity of molecular diagnosis. One of the most widely used genetic markers is the 18S ribosomal RNA (18S rRNA) gene, also known as the small subunit ribosomal RNA gene. It contains conserved and variable regions, allowing it to be used for the detection and identification of many protozoan parasites. The 18S rRNA gene has been used in the molecular diagnosis of parasites such as Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., and Plasmodium spp. Its main advantage is the availability of conserved sequences suitable for primer design, while variable regions can provide information for species identification. For example, species-specific PCR assays targeting 18S rRNA can distinguish E. histolytica from the morphologically similar E. dispar. Another important group of molecular markers is the internal transcribed spacer (ITS) regions, particularly ITS1 and ITS2. These regions are located between ribosomal RNA genes and are generally more variable than 18S rRNA. Consequently, ITS markers are particularly useful for distinguishing closely related parasite species and for molecular epidemiological studies. The ribosomal DNA region can be represented as 18S–ITS1–5.8S–ITS2–28S. The 28S rRNA gene, or large subunit rRNA gene, is also useful for parasite identification and phylogenetic analysis. Mitochondrial genes represent another important category of molecular markers. The most commonly used include cytochrome c oxidase subunit I (COI or cox1) and cytochrome b (cytb). These genes contain considerable sequence variation and are therefore useful for species identification, molecular barcoding, and genetic characterization. Mitochondrial markers are particularly valuable for differentiating closely related species of helminths and protozoa and for studying parasite populations. Some parasites possess highly repetitive DNA sequences that provide extremely sensitive targets for PCR. A well-known example is Toxoplasma gondii. Two important molecular targets are the B1 gene and the 529-bp repetitive DNA element. The 529-bp repeat occurs in hundreds of copies in the parasite genome, making it an excellent target when the parasite burden is very low. It is therefore widely used in sensitive PCR and qPCR assays, particularly for the detection of T. gondii DNA in clinical specimens. Other parasites have specific genetic targets that are particularly useful for molecular diagnosis. In Giardia duodenalis, genes such as β-giardin, glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) are commonly used for molecular characterization and differentiation of assemblages. In Cryptosporidium spp., the 18S rRNA, COWP, and gp60 genes are important targets, with gp60 being particularly useful for subtype identification. For Leishmania and Trypanosoma, kinetoplast DNA (kDNA) is an important target because of its high copy number, which can improve the sensitivity of molecular detection. The choice of a molecular target therefore depends on the purpose of the diagnosis. Conserved genes such as 18S rRNA are useful for broad parasite detection and taxonomic identification, whereas more variable regions such as ITS and mitochondrial COI/cytb are more suitable for species differentiation and genotyping.