We conducted a 2-y investigation to assess the efficacy of trail cameras to identify individual Puma concolor coryi (Florida Panther). We established 35 camera sites within the 28,328-ha northern Addition Lands region of Big Cypress National Preserve from 1 January 2011 to 31 December 2012. To maximize the number of Florida Panthers captured, we intentionally avoided the use of transects or grids for camera-site selection. Instead, we placed cameras along known Florida Panther travel routes. We used a scent lure at each camera site to encourage Florida Panthers to linger in camera range, thereby increasing the opportunity to determine gender and observe anomalies that would aid in identification of individuals. Our cameras captured Florida Panthers 2154 times, which produced a total of 38,056 individual photos. We determined the identity of individual male Florida Panthers in 93% of captures (n = 1190 of 1278). However, the absence of anomalies in adult female Florida Panthers prevented us from identifying them consistently and with absolute certainty, despite thousands of opportunities to do so. Therefore, we relied on the morphological characteristics of dependent kittens to identify individual females in specific instances. We feel that the modifications to the camera survey (i.e., cameras placed on travel routes, high-quality digital cameras, and use of a species-specific scent lure) increased our ability to determine gender and identify individuals.