The genotyping of 25 isolates of Toxoplasma gondii from free-range chickens in the state of Bahia, Brazil, was performed by PCR-restriction fragment length polymorphism using 11 genetic markers: SAG1, 5′ 3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. The analysis revealed 8 genotypes, 3 of which had not been previously reported. Four genotypes were represented by single isolates, whereas the other genotypes were represented by 2 or more isolates. Five isolates showed mixed infections, and 2 of them were identical. None of the clonal types I, II, or III were found, but 2 isolates corresponded to the Brazilian clonal lineage BrIII. There was a single allele for the c22-8 marker. The CS3 marker demonstrated efficiency in the evaluation of virulence in mice. This study reaffirms the diverse genetic variability of T. gondii in Brazil.