Enterolobium cyclocarpum is a characteristic legume tree species of seasonally dry tropical forests (SDTFs) of Mesoamerica and northern South America typically used in silvopastoral and agroforestry systems. Remaining populations of E. cyclocarpum in Colombia are severely fragmented owing to the highly degraded state of SDTF in the country, posing threats to both their in situ persistence and their usefulness as seed sources for future planting efforts. We genotyped E. cyclocarpum populations at nine sampling sites across a latitudinal gradient of SDTF in Colombia by means of eight nSSR markers to elucidate the species diversity distribution in the country. Our data suggest that a deep divide seems to have existed between Caribbean and Andean populations of E. cyclocarpum in Colombian SDTF that may date back to the last glacial maximum (~21,000 BP), or longer. However, we only found evidence of genetic differentiation between trees from the southern Cauca River valley and populations at more northern locations. All the latter populations showed signs of admixture which may be the result of human-influenced movement of germplasm, particularly after the introduction of cattle by European settlers. Most of the sampled sites showed heterozygosity scores close to Hardy–Weinberg expectations. Only the three southern-most populations displayed significantly positive values of inbreeding coefficient, potentially affecting their in situ maintenance and their use as seed sources. Based on our findings we identify priority areas for the in situ conservation of remaining E. cyclocarpum populations, and propose a strategy for sourcing of appropriate planting material for use in future tree planting efforts.