Smalltooth sawfish Pristis pectinata use southwest Florida nurseries during at least their first 2 to 3 yr, and understanding region-specific habitat use patterns is important for effective management. Research occurred in 2 nurseries within the Charlotte Harbor estuarine system: the Caloosahatchee River, a highly human-altered nursery, and a more natural nursery, the Peace River. Between 2010 and 2013, a total of 148 juveniles ranging from 70.8 to 218.7 cm stretch total length were caught in gill nets, and 133 were acoustically tagged. The probability of encountering a sawfish during sampling was most influenced by season in both nurseries (highest during spring and summer), followed by dissolved oxygen in the Caloosahatchee River, and salinity and temperature in the Peace River. Temperature had lasting, atypical effects on movements when severe cold events occurred during the study. Outside of these disturbances, sawfish used all habitats available to them in both nurseries, but tended to reside in perennial high-use areas called nursery hotspots. Acoustic monitoring showed that juveniles gradually moved between 4 hotspots along a 20 river kilometer stretch of the Caloosahatchee River with seasonal changes in freshwater inflows or sometimes suddenly in response to large flow events. In contrast, sawfish in the Peace River remained associated with one nursery hotspot year-round. Reasons for these inter-nursery variations are attributed to differences in geomorphology and freshwater inflow regimes. This study illustrates that a full understanding of the life history of a species and the development of a conceptual model requires investigation of the species on a broad spatial scale. This consideration is important to avoid overgeneralization of habitat use patterns between nurseries.