The selection of Jatropha based on morphological information and molecular markers is essential as it is more reliable and consistent. Hence, twelve Jatropha accessions from different geographical areas of India were screened for genetic diversity using 19 morphological traits and 21 ISSR primers. The analysis of morphological traits grouped the accessions into five clusters. The cluster I consisted of J. curcas (CJC 18), J. curcas (CJC 20), J. curcas (CJC 22), J. curcas (CJC21), and J. curcas (CJC 25), and contained the maximum number of accessions; clusters II and IV contained the minimum number of accessions. Among all the characters, the highest range was exhibited by plant height and the least value by the number of branches. The twenty-one ISSR primers generated 156 polymorphic alleles. The average number of ISSR alleles generated was 7.47 per primer. The ISSR primer UBC 884 was highly informative with the maximum of 12 alleles. The 12 genotypes were grouped into eight clusters. The cluster I contained the maximum number of accessions, namely J. curcas (CJC 18), J. curcas (CJC 20), J. curcas (CJC 22), J. curcas (CJC21), and J. curcas (CJC 25). The clusters II, III, IV, V, VI, VII, and VIII (J. tanjorensis, J. gossypiifolia, J. glandulifera, J. podagrica, J. ramanadensis J. villosa, and J. integerrima) contained the minimum number of accessions. Maximum diversity between J. villosa and J. integerrima was noticed and the least diversity between J. curcas (CJC21) and J. curcas (CJC 25) seen because the ISSR markers differentiated the Jatropha accession into a wide genetic diversity as compared to the morphological data. The species-specific diagnostic markers identified in the study such as 1000 bp alleles for J. glandulifera by the primer UBC 826 is suitable for discriminating species of Jatropha, and thus can be used for identifying a Jatropha species from any mixed population comprising other members of the Jatropha complex.