Bat guano is a noninvasive, data-rich genetic resource. However, the constituent bat DNA is relatively scant, degraded, and complexed with polymerase chain reaction inhibitors. It also is comingled with a rich pool of nontarget DNA from microbes, parasites, and dietary items. We designed and tested new DNA assays for bat species identification (COX1-Bat) and sex identification (XGXYC) for use with guano and other challenging samples. We reviewed previously published assays that can be used with guano samples to obtain the same species and sex data, and attempted to validate these assays for species in which they had not previously been tested. Our results demonstrate that guano-derived DNA can be used successfully to 1) identify nearly all US and Canadian bats at the species level, or to one of three Myotis species clusters, and 2) identify the sex of at least 23 US and Canadian bat species. Our newly developed assays, and validation of previously published assays, for guano-based identification of species and sex in bats, significantly enhance the power of noninvasive sampling and genetic analysis for bat studies, management, and conservation.