Reduced recruitment of yellow perch has been noted for a number of years in certain urbanized watersheds (South and Severn Rivers) of the Chesapeake Bay. Other rapidly developing watersheds such as Mattawoman Creek are more recently showing evidence of reduced recruitment of anadromous fishes. In this study, we used a battery of biomarkers to better document the reproductive health of adult yellow perch collected during spring spawning in 2007–2009. Perch were collected in the South and Severn Rivers, Mattawoman Creek and the less developed Choptank and Allen's Fresh watersheds for comparison. Gonadosomatic indices, plasma reproductive hormone concentrations, plasma vitellogenin concentrations and gonad histology were evaluated in mature perch of both sexes. In addition, sperm quantity (cell counts) and quality (total and progressive motility, spermatogenic stage and DNA integrity), were measured in male perch. Many of these biomarkers varied annually and spatially, with some interesting statistical results and trends. Male perch from the Choptank and Allen's Fresh had generally higher sperm counts. In 2008 counts were significantly lower in the perch from the Severn when compared to other sites. The major microscopic gonadal abnormality in males was the proliferation of putative Leydig cells, observed in testes from Severn and less commonly, Mattawoman Creek perch. Observations that could significantly impact egg viability were an apparent lack of final maturation, abnormal yolk and thin, irregular zona pellucida. These were observed primarily in ovaries from Severn, South and less commonly Mattawoman Creek perch. The potential association of these observations with urbanization, impervious surface and chemical contaminants is discussed.