Spotted seatrout (Cynoscion nebulosus) are estuarine residents that have been extensively studied primarily due to their economic importance. Although spotted seatrout have a documented affinity for seagrass beds, there is little information on how seagrass bed architecture, seagrass species composition, and freshwater inflow affect this relationship. The objective of this study was to document patterns in spatiotemporal distribution and abundance of juvenile spotted seatrout (≤100 mm SL) in estuarine seagrass beds. We analyzed long-term, fisheries-independent monitoring data (1996–2009) from Tampa Bay, Florida, and also conducted a short-term, synoptic study specifically targeting seagrass beds with varying vegetative characteristics and degrees of freshwater influence. Juvenile spotted seatrout were most abundant in seagrass beds influenced by freshwater and dominated by shoal grass, Halodule wrightii. Annual abundance varied over the sampling period, with some peaks in juvenile abundance corresponding with high levels of freshwater inflow. Although analysis of long-term data indicated that juvenile spotted seatrout abundance was positively related to estimated percentage cover of seagrass, the canopy cover of seagrass beds measured quantitatively during the synoptic study was less in areas influenced by freshwater. The value of seagrass as spotted seatrout habitat transcends the simple presence or absence of vegetation and can differ based on factors such as location within the bay, seagrass species composition, and amount of freshwater influence, and management decisions regarding this species and the restoration of particular seagrass beds need to be made with full consideration of these complex relationships.