The continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time, the chromosomal analysis, the bacteria, fungi, yeast, and mycoplasma lack of contaminations, and the efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors were investigated and reported. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination. The significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.