Scrophularia ningpoensis Hemsl. is an important Chinese medicinal herb with a history of domestication of over 1000 years. Phytochemical variation of S. ningpoensis in response to environmental gradients remains an attractive topic with both practiceal and theoretical significances. In the current study, HPLC fingerprinting and four major bioactive compounds of S. ningpoensis, that is, harpagoside, angroside C, acteoside, and cinnamic acid, were determined to explore its correlations with climatic, geographic, and soil factors. The present data confirmed the approximate three-group pattern of phytochemical differentiation among the five production regions, the population of Zhejiang (ZJ), the population of Hubei (HB), and the rest three populations of Chongqing, Hunan, and Shaanxi (CQ, HN, and SX). Harpagoside, the dominant bioactive compound of S. ningpoensis, contributed most to the phytochemical differentiation and displayed a significant positive correlation with monthly and annual average temperature and negative correlations with altitude and latitude. It was concluded that harpagoside variation was strongly positively correlated with environmental changes of temperature.