The genetic manipulation of spermatogonial stem cells (SSCs) can be used as an alternative to somatic cell nuclear transfer method for the production of transgenic animals. SSCs are now in vitro cultured and transplanted in sheep, however, there are no known protocol for DNA transfection of sheep SSCs. The aim of present study was to define the optimal transfection conditions of spermatogonial stem cells (SSCs) in early and late ovine SSC colony formation stages in culture. SSCs were isolated from the slaughterhouse ram testis tissue using a two-step enzymatic digestion process. Results showed that, 2 µl of DNA with 0.5 µg of Lipofectamin or Turbofect were able to transfect SSC colonies at late stage. Since the colonies of SSCs were not in logarithmic growth phase, around 15% of colonies were transfected and no significant difference between Lipofectamin or Turbofect was observed. However, more cells were transfected on early stages of SSC colony formation (7th days), especially when Turbofect was used (around 40 and 45% for Lipofectamin and Turbofect, respectively). Although the early stages SSCs were more suitable for transfection, but the formation of colonies were impaired on transfected cells.