Ciliate protozoa of the genus Balantioides can parasitize a variety of animals. The morphology of the evolutionary forms of the parasite and the host species affected have long been the only characteristics used to taxonomically identify the species of these protozoa, but these variables are not very precise. To confirm species identity, molecular biology tools are currently used. In this context, this study aimed to analyze protozoan isolates maintained in culture medium and from fecal samples from captive animals in Rio de Janeiro, Brazil, by means of molecular tools. Forty isolates maintained in Pavlova modified medium (30 were isolated from feces of pigs and 10 from feces of cynomolgus macaques) were analyzed. In addition, 34 fecal samples (8 from pigs, 8 from cynomolgus macaques and 18 from rhesus macaques) containing Balantioides coli-like cysts were analyzed. All samples were subjected to DNA extraction and the polymerase chain reaction (PCR) to amplify the fragment ITS1 - 5.8s rRNA - ITS2, and the PCR products were purified and sequenced. All samples (100%) presented sequences that were grouped in the Balantioides coli cluster. The type A0 variant predominated. These sequences were 96% to 99% identical to those deposited in GenBank, including a B. coli sequence that had been obtained from human fecal material in Bolivia. It seems that the culturing system did not select variants, because this variant was also seen in the amplified sequences of fecal samples containing cysts. The isolate sequences in the cultures showed few ambiguities and substitutions, thus generating reliable chromatograms. This was the first study to identify B. coli in captive animals in Brazil, through molecular biology. In addition, it was the first to evaluate a large panel of isolates of the parasite through culturing.