Canine influenza virus (CIV) emerged around 2000 when an equine influenza virus (EIV) was transmitted to dogs in Florida. After 2003, the canine virus was carried by infected greyhounds to various parts of the United States and then became established in several large animal shelters, where it has continued to circulate. To better understand the evolution of CIV since its emergence, and particularly its microevolution in spatially restricted populations, we examined multiple gene segments of CIV from dogs resident in two large animal shelters in New York City during the period 2006 to 2009. In particular, we focused on viruses circulating in the two shelters in 2008 and 2009, which we found shared a common ancestor. While viruses in each shelter were generally monophyletic, we observed some gene flow between them. These shelter sequences were compared to earlier CIV isolates. The shelter viruses differed in 1 to 6 amino acids in each gene segment compared to viruses isolated in Florida between 2003 and 2005 and in Colorado in 2006 and 2008. A comparison of the sequences of equine and canine viruses revealed amino acid replacements that distinguished the viruses from the two hosts, but no clear evidence of positive selection indicative of host adaptation was detected, suggesting that any host range adaptation in CIV occurred early in the emergence of this virus or even before it transferred to dogs.