The increased cost of biodiesel production from edible oils has motivated the researchers to find alternative non-edible oil feedstocks. Jatropha curcas has been identified as a promising tropical and subtropical perennial energy crop for biodiesel production. However, securing adequate J. curcas oil for sustainable biodiesel production is not achieved yet. Therefore, the present study sought to investigate the potential and suitability of J. curcas grown at different areas of Ethiopia for biodiesel production using analytical, instrumental, and empirical methods. The dry weight proportion of J. curcas seeds, seed coats, and kernels were significantly varied across growing ecologies. The oil contents of kernels were reneged between 47.10–59.32%, while the free fatty acid content of all sampled oils was less than 2.7%. The estimated iodine and saponification values were ranged between 99.6–112.6 mg I2 and 180.9–202.0 mg KOH per gram of oil, respectively. Oleic (34.2–42.2%) and Linoleic (34.8–41.8%) acids dominated the fatty acid composition of J. curcas oil. The biodiesel fuel properties such as kinematic viscosity, density, cold filter plugging point, and cetane numbers predicted from the fatty acid composition of oils were agreed with EN-14214 standards. The k-means cluster revealed that kernels harvested from different geographic locations were grouped in the same cluster, which indicates that the effect of altitudinal variation on the oil content of J. curcas was not significant. However, at too lower and higher altitudes, the kernel’s oil content has significantly declined. In conclusion, Ethiopian variety J. curcas oil showed suitable physicochemical properties for biodiesel production. However, its yield, composition, and physicochemical properties were significantly varied across growing areas.