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Introduction Rhodococcus equi (R. equi) is considered one of the most economically significant diseases in the equine industry, causing bronchopneumonia in foals. Pili are typically associated with adhesions to cells or the movement of genetic material between bacterial cells. R. equi pili may play roles in immunorecognition and virulence, but their actual function is unknown. Here we describe the first molecular investigation into the presence and variability that exists in virulent R. equi pilus determinants. Materials and methods This study investigated 45 virulent R. equi isolates from foals with pneumonia across NSW and Victoria. PCR protocols were developed to detect and genetically characterise the RplB pilin subunit gene. PCR products were genetically sequenced and compared to detect variation in the RplB gene. Fisher’s exact test was used to explore any associations between pilus alleles and farm or strain type. Results Four pilus subtypes were determined based on their allelic variations in the hydrophobic domain of the pilin subunit. Farms often had multiple pilus subtype strains present, and there was a statistical association (P < 0.05) between prominent pilus subtypes and restriction fragment length polymorphism (RFLP) R. equi strain types. Relevance to Australian clinical equine practice The relevance of pilus variability in circulating strains of virulent R. equi on Australian stud farms cannot be determined, as the biological and pathological significance of R. equi pili have yet to be determined. However, the diversity in the cytoadhesive R. equi pilus must be considered if pilus-based vaccine strategies are to be developed in the future. [ABSTRACT FROM AUTHOR]