Biofilm formation in Staphylococcus aureus is considered an important virulence factor in bovine mastitis. Intercellular adhesion gene A (icaA) is a significant genetic determinant that contributes in biofilm formation. The aim of the present study was to determine the presence of the icaA gene in S. aureus isolated from bovine mastitis from seven states of India. A total of 88 out of 150 Staphylococcus aureus strains were found to be positive for biofilm marker icaA gene by PCR. The icaA gene was confirmed by dot blot hybridization in 41 of 150 S. aureus strains tested. Results obtained with dot blot hybridization were comparable to those obtained with PCR. Partial sequences of the icaA gene of the two S. aureus isolates showed deletion of some bases in different positions that might reduce/stop transcription leading to no biofilm formation. PCR was found to be a rapid test but dot blot hybridization was more accurate than PCR for detection of icaA genes. This study showed that detection of biofilm marker the icaA gene in S. aureus would allow the detection of virulence factors present in mastitis and early application of corrective measures.