Spatial variability of soil, land use and topographic features along stream network were analyzed in a fifth order agricultural watershed of East-central Pennsylvania. The calculated ratios of bifurcation, length and area were in the general range of the Horton’ laws. Mean elevation, slope and agricultural land use in different order sub-watersheds were influenced by topographic characteristics in the Valley and Ridge Province. The ridge distributed in the higher order sub-watersheds led to the relative higher elevation and slope and lower agricultural land use. Limited variability of six soil properties (depth to bedrock, total available water capacity, total clay content, bulk density and saturated hydraulic conductivity) were observed among different order sub-watersheds. However, a series of trends of soil properties were detected along six buffer zones of different order streams. Water quality variables including nitrate, chloride and sulfate were investigated for 31 sites located different order streams during two years’ baseflow. Nitrate and chloride concentration showed a decreasing trend with increasing stream order. Nitrate concentration can be also linearly correlated to agricultural land use. It was concluded that variability analysis of landscape and water quality along stream network would assist in understanding NPS pollutant transport and improving their modeling in agricultural watersheds.