The Florida manatee Trichechus manatus latirostris occupies coastal and riverine habitats that may influence the species’ endogenous biological rhythms, including its reproductive potential. Inhibin B provides a biomarker of gonadal function and reproductive potential in humans and other eutherian mammals. This study examined the influence of size, sex, and time of year on inhibin B levels in manatees sampled among 3 habitats with varying degrees of environmental stress in Florida. Inhibin B levels in 38 males averaged (±SE) 4.90 ± 0.23 pg ml^-1; the average level in 31 females was 5.63 ± 0.46 pg ml^-1. Elevated patterns in inhibin B were exhibited between mid-March and mid-August corresponding to increased mating activity and testicular function, with significant differences in inhibin B levels between male and female manatees (p = 0.03) throughout the year. No significant differences in inhibin B were detected between low- and high-impacted sampling locations during winter, suggesting the potential influence of environmental stress on manatee reproduction may be best examined between mid-March and mid-August—the midpoint of the reproductively active, non-winter time period. Establishing temporal baselines for inhibin B values may be useful in assessing manatee reproductive status and potential conservation threats, shedding light on fertility potential, and enabling future assessment of the effects of stressors on reproduction in Florida manatees.