We tested the utility of a protocol using genetic markers that previously proved successful to identify the sex of Vespertilionid bats on tissues collected from live bats and carcasses of varying age from the Hawaiian hoary bat (Lasiurus cinereus semotus). This molecular method is based on genes unique to X and Y chromosomes in mammals and previously was used successfully on North American hoary bats (L. c. cinereus). We amplified two markers within intron regions of the zinc-finger-X (Zfx) and zinc-finger-Y (Zfy) genes using a multiplexed polymerase chain reaction technique and obtained product bands that were easily visualized using gel electrophoresis. Genotyping determined the sex of 36 individual Hawaiian hoary bat carcasses previously assigned sex only by external genitalia and identified sex for 29 “unknown” bat carcasses that could not be classified by external genitalia. Employing this method for sexing Hawaiian hoary bats will permit more reliable evaluation of the ratio of males to females in subpopulations affected by fatalities from emerging threats. This is critical to the conservation and management of this endangered bat.