Amplified fragment length polymorphism (AFLP) analysis was used to determine genetic relationships among 32 accessions of the species Stylosanthes humilis, representing 26 and 6 accessions of Mexican and South American origin, respectively. Seven AFLP primer pairs generated a total of 111 polymorphic bands with an average of 15.8 polymorphisms per combination and a polymorphic information content ranging from 0.89 to 0.79. Cluster analysis and principal coordinates analysis of the calculated similarity matrix clearly separated all the Mexican accessions, with one exception, from the accessions of South American origin. The observation of differentiation of these two main groups by AFLP analysis is highly congruent with previous studies based on morphological descriptors, cross-hybridization experiments, and chloroplast DNA sequence analysis, and may suggest that both groups evolved separately due to geographical isolation in the past, resulting in the existence of at least 2 principal gene pools. Within the Mexican collection, three groups were recognized, each corresponding to a geographical region with a low within-group and a high between-group genetic variability. Although most Mexican accessions formed one major group, one accession clustered with the South American gene pool. These findings show that Mexico may contain unique sources of S. humilis and therefore would merit attention for conservation and maintenance of S. humilis germplasm. Also, these results demonstrate that AFLP analysis is an efficient method for assessing genetic diversity among S. humilis accessions.