Stylosanthes guianenesis Sw. is an important tropical forage legume grown and utilized in the tropics and the subtropics of South China. Anthracnose, caused by Colletotrichum gloeosporioides (Penz.) Sacc., is a major constraint to the extensive use of Stylosanthes. Forty-five accessions of S. guianensis were assessed with RAPD for genetic diversity and for resistance to anthracnose. RAPD analysis was performed using twenty primers screened from 200 arbitrary oligonucleotides, and a 71.5% level of polymorphism was found. The dendrogram obtained with unweighted pair group method of averages (UPGMA) based on the RAPD data showed genetic similarity from 50% to 94% among all stylo accessions, and fourteen clusters were defined at 66.5% genetic similarity. Two strains of C. gloeosporioides from stylo in China were used for anthracnose resistance screening. All plant accessions showed variation in the reaction to two strains and the correlation of resistance had a value of 0.904. Multiple correspondence analysis displayed a random distribution of the resistance or susceptibility response with respect to the genetic diversity measured by RAPD analysis except one group. Mean distance was also calculated to determine the diversity within clusters. From our results, the RAPD analysis is an effective and efficient technique of providing quantitative estimates of genetic similarity among stylo accessions.