Headwater streams dominate natural landscapes and provide essential functions for downstream waters. However, because of minimal legal protection, they often are piped or buried to accommodate urban growth. Urbanization also alters stream base flows. The combined impact of these factors on channel location is unknown. We assessed the effects of urbanization on the location and length of ephemeral, intermittent, and perennial streams. We randomly selected 150 of 6686 potential channel origins in Hamilton County (Cincinnati), Ohio, USA, for field assessments, and mapped 122 ephemeral, 74 intermittent, and 45 perennial flow origins in these channels. On average, 1:100,000- and 1:24,000-scale US Geological Survey maps underestimated channel length by 85% and 78%, respectively. Mean catchment areas for ephemeral and intermittent flow origins were smaller in forested (0.66 ha and 3.60 ha, respectively) than in urban areas (5.13 ha and 6.79 ha, respectively). These values indicate 93% and 46% county-wide losses of ephemeral and intermittent channel length, respectively, with urbanization. In contrast, the mean catchment area for perennial flow origins was larger in forested (48.12 ha) than in urban (31.22 ha) areas, resulting in a 22% gain in perennial channel length with urban development. Increased perennial channel length was partially explained by reduced forest cover, a result suggesting that reduced evapotranspiration can significantly increase stream base flows. Most variation (59–74%) in catchment area of ephemeral, intermittent, and perennial flow origins was explained by catchment relief, with higher relief corresponding to smaller catchments. Urbanization can decrease (e.g., via reduced infiltration) or increase (e.g., via lawn irrigation and septic tanks) the permanence of flows, thus confounding any overall effect of urban land cover on hydrologic permanence. Site-specific differences in physiography (e.g., bedrock, springs) and landscape management (e.g., stream impoundments) suggest that field surveys are necessary for accurate stream delineation. These results highlight the extensive effects of urbanization on the presence and hydrologic permanence of headwater streams, raise issues with current jurisdictional policy in the US, and emphasize the need to examine the cumulative effects of headwater stream loss on downstream ecosystems.