A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five genome regions. Of these, 62 isolates were HAdV-B3 and HAdV-B7 collected during a simultaneous 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 and other species B adenovirus types collected in different years following the outbreak. The phylogenetic analysis results of all the isolates in the structural regions hexon L2, penton, and fiber knob were found to be consistent and no mismatches were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable even when different types are circulating in a limited geographical area simultaneously. J. Med. Virol. 87: 117–124, 2015. © 2014 Wiley Periodicals, Inc.