Abstract: Milk and milk products have been used by human population since ancient ages and have been a well known source of lactobacilli. The present study is directed towards the study of prevalence, isolation and identification of Lactobacillus species in milk. Milk samples were collected from the cow and buffalo sheds of a local dairy in the Aarey Milk Colony which is a major supplier to the city of Mumbai. A total of 40 milk samples were obtained and 163 colonies were isolated from them. These colonies were subjected to characterization by standard microbiological methods. On the basis of physiological tests and sugar utilization pattern, all the 163 isolates were confirmed to belong to the genus Lactobacillus: L.fermentum (48%), L.acidophilus (34%), L.viridescens (8%), L.brevis (5%), L.gasseri (4%) whereas two isolates could not be identified upto the species level. The results indicate that L.fermentum is predominant in the milk obtained from this sector.