Fasciolosis caused by Fasciola hepatica is a common parasitic disease of livestock especially sheep and cattle. In this study molecular characterization of β-tubulin isotype 3 gene in Fasciola hepatica isolates from cattle and sheep in Turkey was carried out. For this purpose a total of 80 adult Fasciola hepatica isolates were collected from 20 sheep and 20 cattle in Kayseri and Erzurum provinces. PCR-RFLP was performed on β-tubulin isotype 3 gene and MboII revealed two fragments of approximately 350 bp and 390 bp, whereas HphI enzyme yielded 210, 340 and 540 bp bands, HindII yielded 380 and 450 bp bands in all samples. A total of 80 isolates were tested by SSCP and all of them presented the same band profiles. Six samples (4 sheep and 2 cattle) were randomly selected and DNA sequence of a 935 bp coding fragment of β-tubulin isotype 3 was performed. Sheep samples were more polymorphic than the cattle. This β-tubulin isotype 3 gene polymorphism of F.hepatica isolates from sheep and cattle of two distinct geographical areas of Turkey have been investigated for the first time.