Technical Abstract: Although onion is a globally significant vegetable crop, the structure of genetic variation within and among populations is poorly understood. We broadly surveyed allelic variation in cultivated onion germplasm by genotyping population samples from 90 landrace, inbred and open-pollinated varieties of wide geographical adaptation with 69 EST-SSR and 13 genomic SSR markers. EST-SSRs revealed limited allelic diversity but high heterozygosity. Re-sequencing of 25 marker products revealed additional SNPs in 17 (68%), suggesting that mutation scanning of 5’UTR amplicons from ESTs may be a more productive approach to EST marker development than electronic methods. A significant proportion of the EST primer sets amplified products in A. roylei (84%), A. fistulosum (83%) and shallot (99%), showing they can be readily used for comparative and interspecific studies. Di-nucleotide genomic SSRs were more highly polymorphic than EST-SSRs but were less suitable for genotyping population samples. Multivariate analysis of genetic distances among populations revealed genetic relationships consistent with known ancestry and origin, in particular resolving long-day and short-day populations and populations from the Indian subcontinent. Landrace and open-pollinated populations exhibited higher levels of gene diversity than landrace accessions, suggesting that widely cultivated open-pollinated varieties are a key reservoir of genetic variation in bulb onion.