To determine whether Powassan virus (POW) and deer tick virus (DTV) constit
ute distinct flaviviral populations transmitted by ixodid ticks in North Am
erica, we analysed diverse nucleotide sequences from 16 strains of these vi
ruses. Two distinct genetic lineages are evident, which may be defined by g
eographical and host associations. The nucleotide and amino acid sequences
of lineage one (comprising New York and Canadian POW isolates) are highly c
onserved across time and space, but those of lineage two (comprising isolat
es from deer ticks and a fox) are more variable. The divergence between lin
eages is much greater than the variation within either lineage, and lineage
two appears to be more diverse genetically than is lineage one. Applicatio
n of McDonald-Kreitman tests to the sequences of these strains indicates th
at adaptive evolution of the envelope protein separates lineage one from li
neage two. The two POW lineages circulating in North America possess a patt
ern of genetic diversity suggesting that they comprise distinct subtypes th
at may perpetuate in separate enzootic cycles.