Prevalence levels of LaCrosse (LAC) virus in larvae from overwintered eggs of Aedes triseriatus were between .0029 and .0059 (calculated as minimum field infection rates) during a 2-year study of 2 localities in south-western Wisconsin that have long been known as sites of endemic LAC virus activity. In several other localities that had not been previously studied but which had ample Ae. triseriatus populations overwintering virus prevalence did not exceed .0014. In a one-year comparison of a north slope with an opposing south slope that was drier, had less ground cover and fewer chipmunks, a significantly higher overwintering virus prevalence was detected on the north slope. It appeared that within a given endemic locality, any treehole capable of supporting the development of Ae. triseriatus larvae is a potential overwintering site for LAC virus. The data provide baseline information on virus prevalence that will be useful in attempting to model the epidemiology of LAC virus.