The objective of the present study was to characterise Indian isolates of Mycobacterium avium subspecies paratuberculosis (MAP) by pulsed-field gel electrophoresis (PFGE) and IS900 PCR. This technique has proved to be effective in detecting more polymorphisms than IS900-RFLP. In the present study, strains of MAP isolated from naturally occurring paratuberculosis infection in cattle (5) and goat (4) were characterised by PFGE. Isolates were successfully grown in Middlebrook 7H9 broth with ADC supplement and mycobactin J. All strains were found to have IS900 and F57 genes by PCR. PFGE analysis with Sna B1 revealed that the profiles of the cattle and goat isolates were identical and had a unique profile when compared with profiles currently in the PFGE database. PFGE analysis with Spe 1 demonstrated that all of the isolates were identical to SpeI profile 1 in the database. Studies are continuing to type the isolates by restriction fragment length polymorphism analysis. It was concluded that Indian isolates from cattle and goats were genetically similar but different from European strains. This study provides useful data that ultimately will facilitate control measures for the disease in India.