Although the presence of endosymbiotic bacteria—specifically Rickettsia—have been reported in the ciliated protozoan Ichthyophthirius multifiliis, an obligate parasite that infects freshwater fish, it remains unclear whether these bacteria are present in most, if not all, isolates of I. multifiliis. In this study we report identification of these endosymbiotic bacteria in 18 different isolates of I. multifiliis, from three distinct geographical regions worldwide (Brazil, Taiwan, and United States). In three isolates (Ark11, Ark12, and G15) the presence of endosymbiotic bacteria were determined by deep sequencing, and in the remaining 15 isolates bacteria were detected by PCR amplification of conserved regions of the bacterial 16S rDNA genes. All Ichthyophthirius isolates appear to contain at least one rickettsial rDNA sequence. This ubiquity suggests that I. multifiliis—which has a very reduced genome— may be dependent on this endosymbiotic relationship. Results derived from phylogenetic analyses based on rickettsial rDNA sequences show that the rickettsial bacteria can be clustered into four groups. Some I. multifiliis isolates—particularly those collected from pet stores—were infected by multiple groups of rickettsial bacteria, suggesting that the association between Rickettsia and I. multifiliis is more dynamic than previously thought. Our results also support the hypothesis that endosymbiotic Rickettsia is transmitted both vertically and horizontally, although mechanisms accounted for horizontal transfer remain unknown.