Introduction: There are no reports describing polymorphisms in target genes of anti-Toxoplasma drugs in South American isolates.Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase (dhfr) and dihydropteroate-synthase (dhps) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii.Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced.Results: One polymorphism (A ↔ G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G ↔ C) at positions 259 and 260 and one polymorphism (T ↔ G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations.Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.