Abstract: Forty-one Crotalaria latifolia wild resources in Yunnan were analyzed using inter-simple sequence repeats (ISSR). Sixteen of 100 primers were selected, and 164 DNA fragments were amplified from 41 samples to give 159 fragments (99.3%) that were polymorphic, an average of 10.3 DNA bands per primer. The genetic similarity among all accessions ranged from 0.481 7 to 1.000 0, indicating a comparatively rich genetic diversity among the tested accessions. A better PCR reaction system was obtained using an orthogonal design: DNA 40 ng, primer 0.2 μmol/L, dNTP 200 μmol/L, 1×Buffer (Mg²⁺ plus), Taq polymerase 0.5 U (TaKaRa). The best annealing temperature for ISSR primers was 50 or 53℃. UPGMA cluster based on genetic similarity divided the accessions into five groups, with close intraspecific germplasm. Crotalaria szemoensis was identified by ISSR and its genetic similarity was close to Crotalaria but more work is needed to identify this species.