High-resolution, axial sampling surveys were conducted in Chesapeake Bay during April, July, and October from 1996 to 2000 using a towed sampling device equipped with sensors for depth, temperature, conductivity, oxygen, fluorescence, and an optical plankton counter (OPC). The results suggest that the axial distribution and variability of hydrographic and biological parameters in Chesapeake Bay were primarily influenced by the source and magnitude of freshwater input. Bay-wide spatial trends in the water column-averaged values of salinity were linear functions of distance from the main source of freshwater, the Susquehanna River, at the head of the bay. However, spatial trends in the water column-averaged values of temperature, dissolved oxygen, chlorophyll-a and zooplankton biomass were nonlinear along the axis of the bay. Autocorrelation analysis and the residuals of linear and quadratic regressions between each variable and latitude were used to quantify the patch sizes for each axial transect. The patch sizes of each variable depended on whether the data were detrended, and the detrending techniques applied. However, the patch size of each variable was generally larger using the original data compared to the detrended data. The patch sizes of salinity were larger than those for dissolved oxygen, chlorophyll-a and zooplankton biomass, suggesting that more localized processes influence the production and consumption of plankton. This high-resolution quantification of the zooplankton spatial variability and patch size can be used for more realistic assessments of the zooplankton forage base for larval fish species.