Colombia, situated at the northern end of the Andes mountains of South America and in proximity to Central America, is an important centre of diversity for common bean (Phaseolus vulgaris L.) that has a mix of cultivated germplasm from both major gene pools (Andean and Mesoamerican) for the species. Microsatellites are a useful marker system for analyzing genetic diversity of this crop and can be analyzed with manual (silver-stain) or automated (ABI) detection systems and using unlabelled or fluorescently labelled markers, respectively. The objectives of this research were to evaluate the genetic diversity of 92 Colombian landraces and gene pool controls with 36 fluorescent and 30 non-fluorescent microsatellite markers and to determine the extent of introgression between the Andean and Mesoamerican gene pools for this germplasm. A comparison of fluorescent versus non-fluorescent marker systems was performed with 14 loci, which were evaluated with both methods; the fluorescent markers were found to be more precise than the non-fluorescent markers in determining population structure. A combined analysis of 52 microsatellites using the 36 fluorescent markers and 16 non-overlapping, silver-stained markers produced an accurate population structure for the Andean gene pool that separated race Nueva Granada and race Peru genotypes and clearly identified introgression between these races and the gene pools. The results of this research are important for the application of microsatellite markers to diversity analysis in common bean and for the conservation of landraces in Colombia and neighbouring countries of Latin America, where similar germplasm exists and where gene pool or race mixtures also occur.