Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study， buffalo spermatogonial stem-like cells were isolated， cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia， testes from age 3 to 7 months of buffalo were decapsulated， and seminiferous tubules were enzymatically dissociated. Two types of cells， immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes， OCT3/4 (Pou5f1)， THY-1， c-kit， PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin， were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells， respectively. In the following， when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium， SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells， and accelerating the generation of genetic modified buffaloes.