Gastrointestinal protozoan infections are a leading cause of diarrheal diseases worldwide, particularly in regions with inadequate sanitation and limited access to clean water. This study aimed to molecularly detect and phylogenetically analyze four major protozoan parasites Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, and Blastocystis hominis in stool samples collected from diarrhea patients attending Al-Diwaniyah Hospital in Iraq. A total of 100 stool samples were processed, with DNA extraction performed using the Presto™ Stool DNA Extraction Kit (Geneaid, Taiwan). PCR and nested PCR assays targeting the 18S ribosomal RNA (rRNA) gene were employed for amplification, followed by sequencing and phylogenetic analysis using MEGA X and ClustalW tools. The results revealed a genes of different intestinal parasite species was shown in figure (4-4). The present result showed the 18Sribosomal RNA gene for detection B. hominis were reported in 72 (72.0%) of patients, 12 (12.0%) of patients have G. lamblia infection, 62 (62.0%) have E. histolytica infection and the 18S ribosomal RNA gene for detection C. parvum showed in 34 (34.0%). Phylogenetic analysis demonstrated remarkable genetic conservation among local isolates and global reference strains. For G. intestinalis, the local isolates (IQD.No1–No3) exhibited 99.25–99.65% sequence identity with an Australian reference strain (AF199446.1), with only 0.35–0.75% mutations. Similarly, C. parvum isolates showed 98.85–99.65% identity with an Egyptian reference (AB513881.1), while E. histolytica isolates displayed 99.16–99.45% identity with another Egyptian strain (MK332025.1). B. hominis isolates clustered closely with a Chinese reference (AB197936.1), sharing 99.15–99.71% sequence identity. The UPGMA phylogenetic trees constructed for each species confirmed minimal genetic divergence (0.01%) between local and reference strains. The study underscores the utility of the 18S rRNA gene as a robust molecular