We evaluated the effect of holding temperature and time between mosquito death and processing mosquito pools for virus detection on our ability to detect West Nile (WN) viral RNA from pools of mosquitoes by reverse transcriptase-polymerase chain reaction (RT-PCR). Pools of 24 uninfected Culex pipiens L. mosquitoes were “spiked” with either a single Cx. pipiens that had been inoculated previously with WN virus or with an uninfected mosquito. These pools were held dry at 20, 4, −20, or −70°C for selected time intervals before all mosquito pools were triturated in TRIzol®LS reagent and processed for detection of WN viral RNA. While infectious virus virtually disappeared from pools maintained at 20°C by 48 h after mosquito death, neither holding temperature (20 to −70°C) nor holding period (up to 2 wk) affected detection of WN viral RNA by real-time RT-PCR. These findings suggest that we need not keep mosquitoes chilled to be able to detect WN viral RNA effectively by RT-PCR. This should enhance the feasibility of field-based WN virus surveillance programs where only detection of WN viral RNA is the objective and maintenance of a cold chain may not be possible.