Abstract: Free-living nitrogen fixation (FLNF) by diazotrophs is an important nitrogen (N) source in terrestrial ecosystems and may reprensent a viable solution to environmental pollution caused by N over-fertilization. Studying the impact of different fertilizer regimes in highland barley fields on the diazotrophic community profiles and potential N fixation rates (PNFR) may provide scientific fertilization strategies and a theoretical basis for agricultural green development in the Tibetan Plateau. Here, quantitative PCR, high-throughput sequencing and ¹⁵N labeling methods were used to better understand the impact of different fertilizer regimes on the abundance and composition of diazotrophs as well as the potential N fixation rates in highland barley fields on the Tibetan Plateau. The experiment included five treatments: a control without fertilizer (CK); N, phosphorus (P) and potassium (K) mineral fertilzers (F); manure fertilizer (M); mineral NPK fertilizers plus manure (FM); and mineral NPK fertilizer plus straw (FS). The results showed that: 1) compared with the CK and F treatments, the M, FM and FS treatments significantly (P<0.05) increased the contents of soil organic carbon (C) and total N. Moreover, the ammonium nitrogen (NH₄⁺-N) content was significantly (P<0.05) higher in the FM treatment than in the other treatments. The highest contents of organic C, total N, nitrate N (NO₃⁻-N), available P and available K were observed in the M treatment, and their contents were significantly (P<0.05) higher than those in the other treatments. 2) The PNFR ranged from 2.63 to 4.07 μg∙kg⁻¹∙d⁻¹ under different fertilizer treatments. Fertilization, especially the application of organic fertilizers (sheep manure or straw), reduced the PNFR, and the inhibitory effect of straw on PNFR was higher than that of sheep manure. The soil NH₄⁺-N content was the main factor affecting PNFR. 3) The M and FM treatments significantly (P<0.05) increased diazotrophic abundance, while the opposite was observed for the F and FS treatments. The total N content was the key factor affecting diazotrophic abundance. 4) Different fertilizer patterns significantly changed the diazotrophic composition, and the similarities in the diazotrophic compositions among different fertilizer regimes fell into three major categories: no fertilization (CK), organic fertilization (M), and chemical fertilization (F, FM, FS). Available P was the key factor regulating diazotrophic composition, followed by pH and C/N. In conclusion, the M treatment was the optimal fertilizer practice to improve soil fertility, increase diazotrophic abundance and reduce PNFR decline in highland barley fields on the Tibetan Plateau.