Abstract Introduction Approximately 18% of US Navy sailors are females. Research has shown gender-related differences in the prevalence of sleep disorders in active duty personnel (Foster et al., 2017). Specifically, insomnia, depression, and anxiety are more prevalent in females, while obstructive sleep apnea is more prevalent in males. We have studied the sleep patterns and fatigue levels of crew members on more than 30 US Navy ships. The current study focuses on gender differences in well-being, sleep-related behaviors, and psychomotor vigilance performance of sailors in the US Navy. Methods Using a longitudinal, naturalistic observation paradigm, data were collected from crewmembers on nine USN ships while performing their normal underway duties. Participants (N=1,056) tended to be young (on average 27 years of age), predominantly male (80.6%), and enlisted (84.8%). We assessed average daytime alertness (Epworth Sleepiness Scale), insomnia symptoms (Insomnia Severity Index), mood (Profile of Mood States), and sleep quality (Pittsburgh Sleep Quality Index). Sleep was assessed with actigraphy and logbooks. Sailors performed a 3-minute version of the Psychomotor Vigilance Task (PVT), which was built into their wrist-worn actigraph. Results Compared to males, female sailors reported more depressive symptoms (p=0.042) and less vigor (p<0.001). Females slept more (daily sleep duration: p<0.001) but their sleep was split into more episodes than their male counterparts (p=0.029). Fewer females reported a regular exercise routine (p=0.033). In addition, females report consuming fewer energy drinks (p=0.007), and using fewer nicotine products (p=0.013). Lastly, consistent with findings from civilian populations, female sailors had slower reaction times on the PVT (p<0.001) and experienced more lapses combined with false starts (p<0.001) than their male counterparts. Conclusion Compared to their male peers, female sailors tend to report higher levels of depression and lower levels of vigor. They experience more pronounced split sleep, are less likely to report having an exercise routine, and have poorer performance on the PVT. Fewer females report using energy drinks and nicotine products. Support This research was supported by the Naval Medical Research Center’s Advanced Medical Development Program, the US Navy 21st Century Sailor Office, and the US Navy OPNAV N1.