Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 21 2 2026 07 12 287 296 Faten Fathi College of Basic Education, University of Mosul, Mosul, Iraq Intisar Abdulwahhab College of Education for Women, Tikrit University, Tikrit, Iraq Ahmed Jabar College of Basic Education, University of Mosul, Mosul, Iraq 2025 12 03 2026 07 12 Background: We investigated Dientamoeba fragilis molecular distribution and genetic types among Nineveh Governorate patients with symptoms through SSU rRNA gene PCR amplification and sequencing. Methods: Fifty stool samples were collected from symptomatic patients aged 3–68 years attending the Medical Research Hospital in Nineveh Governorate, northern Iraq in 2024. Genomic DNA was extracted using the Presto™ Stool DNA Extraction Kit. The PCR reaction used species-specific primers DF400/DF1250 to produce ~850 bp amplicons. Positive products were sequenced and examined using BLAST, MEGA 11, DnaSP, and PopART. The Maximum Likelihood method used the Tamura 3-parameter model to perform phylogenetic analysis. PCR detection indicated D. fragilis in 12/50 stool samples, corresponding to a prevalence of 24%. Results: The BLAST analysis showed that the sequence had 97%-99.7% similarity to global reference strains (AY730405). The study identified three haplotypes which contained three mutations while showing a haplotype diversity (Hd) of 0.318 that indicated minimal genetic diversity. The phylogenetic analysis showed that all isolated strains belonged to genotype 1 but U37461 formed a separate lineage as genotype 2. The Iraqi isolates showed sequence similarity to genotype 1 reference strains reported globally. Conclusion: This study provides additional molecular evidence of D. fragilis in northern Iraq and the second report nationally. Genotype 1 was identified among all analyzed isolates with minimal genetic variation between different groups. The molecular detection methods delivered vital diagnostic data but scientists need to expand their surveillance activities to study animal disease transmission to humans and animal disease progression. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/4832 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/4832/1503