Sequencing the onion genome is challenging because of its enormous size (16 giga base pairs DNA per haploid genome). Pilot sequencing of onion transcripts showed sufficient numbers of single nucleotide polymorphisms (SNPs) to develop a detailed genetic map. We used normalized cDNAs from two parental lines of onion: 5225B is a red, doubled-haploid onion and was provided by Seminis Seed Company; OH-1 is a yellow inbred that shows high frequency of gynogenic haploid production. Over 3800 SNPs and 76 indels were identified between the two parents from one and a half 454 plates sequenced at J. Craig Venter Institute. For SNP mapping, a haploid family was generated from the cross of OH-1 with 5225B. Red hybrid bulbs were selected and gynogenic haploids extracted at the University of Ljubljana (Slovenia). DNAs have been extracted from over 185 haploids. These haploid plants have been asexually propagated and evaluation in replicated trials for bulb quality traits without heterozygosity.