In the present study, 120 yeast isolates from different sources (active sludge, soil, and wastewater samples obtained from petroleum refinery and soil contaminated by petroleum) were obtained. The yeast isolates were screened for lipase production and twelve of the isolates (D3, D17, D24, D27, D30, D38, D40, D42, D44, D46, D56, and D57) exhibited lipase activity. Molecular characterization of the yeasts showing the lipase production was performed with RFLP of ITS15.8S- ITS2 and 18S rRNA and sequence analysis of D1/D2 domain of 26S rRNA. The 26S rRNA sequencing revealed that four new strains, D38, D40, D44 and D57 identified as Rhodotorula slooffiae, Candida davisiana, Cryptococcus diffluens, and Cryptococcus uzbekistanensis, respectively, are lipase producing yeast species. This study is the first report showed lipase production by these species. The other lipase producing strains identified as Candida parapsilosis (D3), Rhodotorula muciloginosa (D17 and D42), Cryptococcus albidus (D24, D27, D30, and D56), and Wickerhamomyces anomalus (D46).