West Nile virus (WNV) was introduced into New York in 1999 and subsequently expanded its range to include much of North, Central, and South America. Previously, we have shown that a new strain of WNV (referred to as WN02) that was first detected in 2001 and subsequently spread across North America was more efficient at infecting and being transmitted by Culex mosquitoes than the strain that was originally introduced (referred to as NY99). In the current study, we determined how temperature and time since feeding on infected blood affected the probability that mosquitoes would transmit these two strains of WNV. We found that the advantage of the WN02 strain over the NY99 strain increased with both temperature and time. Thus, warmer temperatures would have facilitated the invasion of the WN02 strain. In addition, we found that transmission of both strains of WNV accelerated sharply with increasing temperature, such that small increases in temperature had relatively large effects on transmission. This laboratory study suggests that both viral evolution and temperature influence the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on virus transmission.