We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an F(o)F(1) ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of "fitness" genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.