In Uruguay, onion (Allium cepa L.) germplasm is mainly derived from the genetic material introduced by several waves of European immigrants and subsequently multiplied by household farmers, resulting in a wealth of locally adapted populations. This study examined the genetic diversity in a collection of 27 local onion populations and two cultivars derived from them. A total of 843 onion plants were fingerprinted, and 83 inter-simple sequence repeat polymorphic bands were generated. Analysis of molecular variance showed high diversity within the populations (66% of the total variation). Some short-day populations from different geographical origins were grouped together by the unweighted pair-group method using arithmetic averages, principal coordinate analysis and cluster analysis, while the more extensively sampled long- and intermediate-day populations showed a widespread distribution, with no significant subgrouping among them. This weakly structured gene pool is probably the consequence of seed and bulb exchange between farmers and natural inter-pollination. Nevertheless, a Bayesian analysis allowed the distinction of four genetic backgrounds of alleles in the whole collection, and populations were predominately assigned to each genetic background. In addition, mitochondrial variants determining normal (N) pollen fertility or the sterile S or T types were analysed for the same set of plants using specific primers. Most accessions showed a proportion of male-sterile individuals. Cytoplasm type T was the most represented (26 out of 29 accessions), and cytoplasm type S was found in a low proportion of individuals in seven populations. Uruguayan onion germplasm maintains a low degree of genetic differentiation despite the small cultivated area and intense seed exchange, probably due in part to different market purposes based on the growing cycle.