Rhodococcus equi is an important pathogen of foals aged 1-6 months. Evidence exists that foals are exposed to a wide diversity of R. equi strains in their environment. However, limited data are available regarding the extent to which genotypic variation exists among isolates infecting individual foals. Therefore, electrophoresis of repetitive sequence-based polymerase chain reaction (rep-PCR) amplicons in an automated microfluidics chip format was used to genotype 9 virulent R. equi isolates obtained from distinct anatomic locations in a single foal. Four of the isolates were obtained from different regions of the lung, and 5 were from abscessed intra-abdominal lymph nodes (LNs). Six distinct genotypes were identified among the 9 isolates. None of the pulmonary isolates was identical; however, a pulmonary isolate was found to be identical to an isolate recovered from a small intestinal LN, and another pulmonary isolate was identical to an isolate from a mesenteric LN. These results indicate that foals can be infected with multiple strains of virulent R. equi. Furthermore, identical strains can be found in multiple, remote anatomic locations in an infected foal, and this can occur for >1 strain in the same foal. The automated system used in the current study provided a rapid, reproducible, and discriminating method for typing R. equi isolates.