Originating in the Welsh Mountains of Lancaster County as a High-Quality – Cold Water Fishery, Pequea Creek generally runs through the study area from northeast to southwest. Upon entering the study area at Route 897, it becomes classified as a Warm Water Fishery, a designation it retains to its mouth at the Susquehanna River.

Two additional waterways are located within the study area: Muddy Run and Houston Run. Muddy Run originated northwest of the study area, is crossed by US 30 and empties directly into Pequea Creek. This waterway is classified as a Warm Water Fishery. Houston Run originates north of the study area and is crossed by US 30 within the eastern section of the study area. Houston Run, which flows directly into the Susquehanna River, is classified as a Cold Water Fishery.

These three primary waterways, Pequea Creek, Muddy Run and Houston Run, were identified in the Route 30 study area. The watershed of these streams cover a combined 135,000 acres, primarily within Lancaster County. The surface waters within the watershed are an important source for drinking water, irrigation, boating, fishing, water sports, wildlife habitat and industrial needs. According to Pennsylvania's Department of Environmental Protection (DEP), 58.5 miles within two watersheds have been degraded by agricultural runoff. Along with pesticide contamination, high concentrations of nitrates, nitrate-nitrogen and ammonia nitrogen have been recorded in surface and ground waters. Cropland is eroding at an alarmingly high rate, increasing the streams' sediment loads. These agricultural impacts have meant that the watersheds are designated as high priority nonpoint source watersheds in Pennsylvania and as a national U.S. Department of Agriculture (USDA) Hydrologic Unit project. Efforts to reduce these impacts are underway through funding and technical assistance provided for by the USDA Water Quality Initiative and provisions documented in sections 319 and 106 of the Clean Water Act.

Mill Creek is located just northwest of the study area. A small portion of its watershed lies with the study area. This waterway is classified as a Warn Water Fishery in the vicinity of the study area, as are its tributaries. It flows into the Conestoga River south of Lancaster, which then flows into the Susquehanna River.