Studying the spatial and temporal patterns of stream fish assemblages and identifying how human land-use activities impact these patterns are crucial for fish species conservation, ecosystem restoration and management. The objectives of this study were (1) to examine the influences of human perturbations on the longitudinal patterns of fish species diversity and assemblage structures in headwater streams, (2) to assess the correlations between local habitat factors and fish assemblages, and their longitudinal and temporal patterns. We sampled fish at 39 segments during May and October 2011 from three headwater streams of the Chencun Reservoir in the Huangshan Area, which were influenced by different extents of human activities. A total of 4041 fish were collected, representing four orders, 10 families, and 28 species, among which family Cypriniformes comprised 67.9% of total species richness. 24, 22 and 24 species were sampled from the Shuxi, Puxi and Maxi Streams, respectively. Two-way ANOVA was used to test the influences of streams and seasons on habitat and fish-diversity variables, respectively. Results showed that habitat variables varied significantly across three streams and in different seasons. Results from the Shuxi Stream showed lower numbers in dissolved oxygen and wetted width but higher levels in substrate size and canopy cover than those from the Puxi and Maxi Streams, and May showed lower pH but higher water temperature and discharge than October. However, species richness, fish abundance and the Shannon's index varied significantly across streams but not between seasons. In overall, fish diversity was higher in the Shuxi Stream than that in the Puxi and Maxi Streams. Results of two-way crossed ANOSIM indicated that fish assemblage structures varied substantially across streams but not between seasons. The assemblage structures in the Puxi and Maxi Streams did not show significant differences, however, they significantly differed from those in the Shuxi Stream. SIMPER analysis indicated that some endemic specialized species (e.g., Zacco platypus, Acrossocheilus fasciatus, Vanmanenia stenosoma) were more abundant in the Shuxi Stream, while those cosmic generalized species (e.g., Misgurnus anguillicaudatus, Pseudorasbora parva, Rhodeus ocellatus) were more in the Puxi and Maxi Streams. The results of Pearson's correlation and redundancy analysis showed that fish species richness in the Shuxi Stream significantly increased with elevation decreasing in both May and October, respectively. However, this "species richness-elevation" correlation lessened in the Maxi Stream and even disappeared in the Puxi Stream. Similarly, elevation showed significant correlation with assemblage structures in the Shuxi and Maxi Streams but not the Puxi Stream. In addition, the effects of local habitat variables on fish assemblage structures were examined using linear regression model and redundancy analysis, respectively. Results showed that fish assemblages in the Shuxi Stream were significantly affected by substrate and canopy, but those in the Puxi and Maxi Streams were related to water temperature and wetted width. In conclusion, our results suggest that, at the sub-watershed scale, the alteration of local habitat conditions in headwater streams associated with human activities (e.g., urbanization, land-use and river-regulation) may decrease stream fish diversity, modify the correlations between local habitat and fish assemblage, and change the longitudinal pattern of fish assemblages.