The flavonoids in bryophytes may have great significance in phylogeny and metabolism research. However, to date there has been little research on bryophyte metabolites, especially flavonoids. To redress this somewhat, we determined flavonoid concentrations of bryophytes from Tianmu Mountain through a colorimetric assay and considered the factors influencing the results. This is the first time that the flavonoid contents of bryophytes have been examined in detail. The results revealed a range of total flavonoid concentrations in 90 samples collected from Tianmu Mountain from 1.8 to 22.3 mg/g (w/w). The total flavonoid contents of liverworts were generally higher than those of mosses; acrocarpous mosses had generally higher values than that of pleurocarpous mosses. The total flavonoid contents of bryophytes growing at lower light levels were general higher than those growing in full-sun. The total flavonoid contents of epiphytic bryophytes were highest, while those of aquatic bryophytes were the lowest. Total flavonoid contents of species growing at low-latitudes were much higher than those at high-latitude individuals. In conclusion, total flavonoid contents of bryophytes have some connection with plant phylogeny; more flavonoids might be contained in relatively primitive bryophytes. Meanwhile, the effects of ecological factors on total flavonoid contents of bryophytes exist; light and habitat (especially tree habitat and river habitat) might be representative factor.