Abstract: After optimization of sterilizing of cyst and larva second stage of Heterodera schachtii, possibility of using nematode on seedlings of sugar beet (Beta vulgaris L.) in in vitro conditions were studied using sterilized larvae of beet cyst nematode. For this purpose, non sterile cysts were extracted from infected soil and hatched into zinc chloride solution with concentration of 0.5 g L-1. Then, for preparation of sterile second stage larvae, several sterilizing treatments were used. Mean comparisons were performed between sterilized live larvae number by Duncan's method. Results showed that 70% ethanol for 1 min followed by 2.5% hypochlorite sodium for 5 minutes and 0.1% hypochlorite sodium for 20 min were best treatments for disinfecting cysts and larvae, respectively. In parallel, two nematode susceptible sugar beet varieties were applied to produce seedlings in in vitro culture. PGoB medium containing different hormonal compositions was used for producing of hairy roots and inoculation of seedling with sterilized larvae. After nematode inoculation tests, daily observations were done by counting cysts and stained roots and larvae under stereomicroscope. Between 5-12 cysts formed on the roots of each seedling from two varieties 40 days after inoculation. As a result, it seems that this technique can be used for sugar beet germplasm evaluation to screen nematode resistant genotypes in in vitro controlled condition.