Alternaria alternata is a well known and economically important seed-borne pathogen with very wide host range. Thirty-eight isolates derived from various hosts from different geographical regions in India including few exotic isolates from Taiwan, Syria and Argentina were used in the study. The molecular characterization using two marker systems i.e. universal rice primers (URP) and inter simple sequence repeat (ISSR) was carried out, which revealed differences based on geographical origin of various A. alternata isolates, which otherwise could not be revealed through conventional characterization. Out of 13 URPs and 20 ISSR primers screened, 5 primers from each marker gave very good reproducible banding patterns. Polymorphism bands ranged between 88.1–100.0% in URP-PCR, whereas it was 100% in case of ISSR-PCR. Maximum heterozygosity (Hn) was revealed by URP 2F and URP 6F (0.29) and least by URP 4R (0.24). In ISSR-PCR, maximum heterozygosity was revealed by ISSR 13 (0.27) followed by ISSR 18 (0.25) and least by ISSR 6 (0.20). Maximum cophenetic correlation was found in ISSR (r = 0.789) followed by URP (r = 0.759). The combined analysis of both marker systems showed high cophenetic correlation (r = 0.810), which indicated a good fit of the data for diversity analysis. The study revealed that to best of our knowledge this is a first report to use of URP-PCR combined with ISSR-PCR and is more sensitive and reliable in characterizing genetic diversity in A. alternata.