Worldwide comparison of Toxoplasma gondii isolates from free-range chickens (Gallus domesticus) has indicated that T. gondii isolates from Brazil are phenotypically and genetically different from isolates from other countries; most strains from Brazil are pathogenic to mice, there is great genetic variability, most isolates are nonclonal, and Type II is absent or rare. The prevalence of T. gondii in 50 free-range chickens from the island of Fernando de Noronha, Brazil (this island is 350 km from the mainland) was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT); 42 (84%) chickens had titers of 1∶5 in 2, 1∶10 in 4, 1∶20 in 3, 1∶40 in 6, 1∶80 in 6, 1∶160 in 5, 1∶320 in 3, and 1∶640 or higher in 13 chickens. Hearts of 40 seropositive chickens were bioassayed individually in mice. Toxoplasma gondii was isolated from 24 chickens with MAT titers of 1∶5 or higher; the isolates were designated TgCKBr210-233. None of the isolates was pathogenic for mice. The restricted fragment length polymorphism using 10 markers revealed 6 genotypes, including the Type II, Type III, and 4 new chicken genotypes (#59–#62) that were different from genotypes so far reported in Brazil. All 24 isolates were successfully genotyped; 15 isolates were Brazil chicken type #59, 1 type #60, 1 type #61, 1 type #62; 5 were Type II (with Type I allele at the Apico locus); and 1 isolate was clonal Type III. Results in this study indicate that T. gondii on this island consists of unique genotypes as well as clonal genotypes that are dominant in Europe and North America.