Background: This study aimed to determine the prevalence of T. gondii infection in humans and sheep in Diyala and Baghdad provinces, Iraq, using serological and molecular techniques, and to genetically characterize circulating strains based on the REGs gene.

Methods: The one hundred placentas of sheep in Diyala and Baghdad ( 50 sample ) from each province were examined. An infected sheep placenta sample was digested by the pepsin enzyme. This is intended for performing the dyeing process with Giemsa stain and extracting the Genomic DNA, depending on the tissue genomic DNA isolation kit ( buffy coat ) of seropositive individuals using Geneaid, Bioteccorp, Taiwan. Polymerase Chain Reaction (PCR) amplification targeted a 529 bp fragment of the REGs gene in Toxoplasma gondii. The human serum sample was collected from 100 human blood samples (male and female) from Diyala and Baghdad ( 50\50). The Enzyme Linked Immunosorbent Assay results were recorded according to the manufacturer’s instructions. the study period is from September 2024 to April 2025.

Results: The overall infection rate in sheep was 25% (25/100), with a higher rate in Diyala (30.3%) than in Baghdad (20%). In ewes, the highest prevalence (43.33%) was observed at 4 years of age. ELISA testing of human sera (n=100) revealed an overall seroprevalence of 23.91%. The rate was significantly higher in Diyala (30.95%) than in Baghdad (18%), and higher in females (40%) than in males (4.76%). The highest infection rate (33.33%) was found in the 25-35-year age group. "Phylogenetic analysis of the Regenerating gene (REGs). gene sequences showed that Iraqi isolates clustered most closely with strains from Iraq, Indonesia, China, Italy, Netherlands, Pakistan, Turkey, Iran, Chile, Myanmar, Nigeria, Saudi Arabia, and India. The phylogenetic analysis of the REGs gene evolutionary distribution of 20 local isolates detected in Diyala and Baghdad (the middle of Iraq), with 36 global isolates of Toxoplasma gondii deposited in GenBank, indicates a diverse evolutionary lineage