Toxoplasma gondii a tissue protozoan affecting one third of humanity has subpopulation structures in different geographical regions caused by less frequent sexual recombination and population sweeps. The majority of strains isolated in North America and Europe fall into one of three clonal lineages, referred to as types I, II, and III. So far, little is known about Toxoplasma strains in Africa. Ocular toxoplasmosis is an important complication of T. gondii infection in the immunocompetent. The present study aimed to determine the genotype of Toxoplasma strains from ocular toxoplasmosis patients in Benghazi, Libya. Blood from 43 patients clinically suspected of ocular toxoplasmosis were obtained and serologically screened for anti-Toxoplasma antibodies. 24 (55.8%) seropositive (IgG) samples were subjected to PCR to obtain T. gondii DNA. Only 6 (25%) samples had enough DNA and were genotyped through single locus nested-PCR-RFLP analysis based upon the SAG2 gene. The restriction pattern revealed the presence of genotype I in all samples. This is the first genotypic evidence (type I strain) based on single locus PCR-RFLP analysis for ocular toxoplasmosis cases from Benghazi, Libya.