Leptospirosis, an environmental spirochete found in fresh water can cause life threatening infections and has caused multiple outbreaks during military operations and training but incidence and impact on Jungle Warfare training is unknown. We conducted a study to estimate the incidence, clinical and mission impact of leptospirosis (both asymptomatic seroconversion and clinical disease) during US military Jungle Warfare Training (JWT) in Hawaii and Okinawa. Following informed consent, risk questionnaires were administered pre and post JWT and an Electronic Medical Record review was conducted for risk factors for infection and to capture clinical cases of Leptospirosis after JWT. Blood was collected before training and both blood and urine collected after training in 1002 participants. We constructed definitions for confirmed and suspected symptomatic and asymptomatic leptospirosis infection. Ninety five percent of participants were students, 79% were aged 18-25, 94% were male and 72% junior enlisted. Ninety-four percent had never taken JWT before. Ninety-four percent completed JWT, 95% were exposed to water, 90% were immersed in water, 57% swallowed water, 26% drank environmental water, 20% unfiltered. 54% reported a skin injury after training. Among 998 pre-training tests, 156 were positive for leptospirosis by ELISA (16%), 30 of these were confirmed by Micro Agglutination Test (MAT). 8 Serovars were detected, Icterohemorrhagicae was the most common. Among 841 post training samples, 0 were positive by PCR, 92 were confirmed by MAT. No patients seroconverted by ELISA and MAT. We had 0 confirmed symptomatic cases and 0 confirmed asymptomatic cases. There were 10 possible asymptomatic cases and 2 possible symptomatic cases. Nine subjects seroconverted by ELISA and 3 by MAT with 2 serovars represented. Seroconversion attack rate was 1.1% in Hawaii and 8.7% in Okinawa. Hawaii does not use doxycycline chemoprophylaxis but in Okinawa 72% took their prophylaxis, 11% missed 1 dose, 6% multiple doses and 5% took no doses. The most common reason for non-adherence was forgetting to take the medication. In summary JWT is a high-risk activity for leptospirosis. Seventeen percent of trainees missed doses of chemoprophylaxis and 5% did not take it at all. Sixteen percent of trainees had evidence of prior leptospirosis exposure before training, this has not been described before and was twofold higher than previous serosurveys on deployed service members. We found 12 probable seroconversions, 2 were symptomatic. Serology did not meet the four-fold increase of our confirmed case definition, however. The lack of symptomatic disease was probably due to chemoprophylaxis. Our observed attack rates were similar to rates in JWT in Panama in the 1980s. Better leptospirosis diagnostic testing is desperately needed.