Here, we investigated benthic diatom assemblages and their seasonal variation in streams along an urban-rural gradient in the Beijing metropolitan area. The samples were collected from 23 sites (7 from rural reference streams, 6 from upstream urban areas, and 10 from downstream urban areas) across three seasons of 2014, namely, spring (May), summer (August), and autumn (November). A total of 200 diatom species and sub-species belonging to 64 genera were identified during the period of sampling. Although Achnanthidium, Cocconeis, Cyclotella, Fragilaria, Gomphonema, Navicula, Nitzschia, Pseudostaurosira, and Staurosira were the dominant genera in the sampling sites, the major dominant genera and species differed considerably among the samples. In rural reference streams, Achnanthidium was the major dominant genus, with an average relative abundance of up to 40.6%. Achnanthidium minutissimum was the major dominant species, with an average relative abundance of 20.51%. In urban upstream sites, Pseudostaurosira and Staurosira were the major dominant genera, with average relative abundances of 21.72% and 22.18%, respectively. Pseudostaurosira brevistriata and Staurosira construens var. venter were the major dominant species, with average relative abundances of 21.72% and 20.17%, respectively. In urban downstream sites, Nitzschia was the major dominant genus, with an average relative abundance of 37.46%, and Nitzschia palea was the major dominant species, with an average relative abundance of 21.7%. There was no significant effect of seasonal variation in the relative abundance of the major dominant genera among the three sampling zones (P > 0.05). The relative abundance of Achnanthidium in reference streams was 34.03% in spring and 37.22% in summer, both of which were lower than that of 43.75% in autumn (P > 0.05). The relative abundances of Pseudostaurosira and Staurosira in urban upstream sites were 19.11% and 23.38% in spring, which were higher than those of 16.79% and 18.78% in summer and 18.18% and 20.49% in autumn (P > 0.05). The relative abundance of Nitzschia in urban downstream sites was 35.71% in spring and 39.27% in summer, both of which were higher than that of 27.25% in autumn (P > 0.05). The relative abundance of the major dominant species did not differ significantly in the three seasons. The relative abundance of A. minutissimum in the reference streams was 18.07% in spring and 17.44% in summer, which were lower than that (25.23%) in autumn (P > 0.05). The relative abundances of P. brevistriata and S. construens var. venter in urban upstream sites were 19.11% and 22.21% in spring, respectively, which were higher than the corresponding values of 16.79% and 16.71% in summer, and 18.18% and 18.86% in autumn (P > 0.05). The relative abundance of N. palea in urban downstream sites was 18.43% in spring and 13.2% in autumn, which were lower than that of 24.74% in summer (P > 0.05). However, the results showed that changes in the relative abundance of Achnanthidium and A. minutissimum in reference streams and Pseudostaurosira and P. brevistriata in urban upstream sites were larger in spring than in autumn and summer. Similarly, changes in the relative abundance of Staurosira and S. construens var. venter in urban upstream sites and Nitzschia and N. palea in urban downstream sites were larger in spring than in summer and autumn. Our results indicate that the distribution of diatom assemblages and their seasonal compositions might well reflect differences in urban-rural stream environments and their changes, and thus the diatom assemblages can be used for biological monitoring of streams in Beijing.