Biological soil crusts (BSCs) are key contributor of carbon sequestration of arid land ecosystem. Carbon-fixing microbes are the critical functional groups in the carbon sequestration in biological soil crusts. However, the detailed research on carbon-fixing microbial diversity of BSCs in Mu Us Sandy Land was not reported. To explore the variation of the abundance and community diversity of autotrophs in the succession of biological soil crusts, qPCR and Micseq sequencing were performed for the samples from Mu Us Sandy Land. The key environmental factors driving carbon-fixing microbial community were also explored. The results indicated that the abundance of form IAB, IC and ID gene increased with the development of BSCs and reached the highest in lichen crusts, then decreased clearly in moss crusts. Chao1 of form IAB and Shannon index of form IC showed the increasing trends with the succession of BSCs, whereas Chao1 of form IC and ID reached the highest in algal crusts. In terms of species composition, IAB-type autotrophic microbes were dominated by Oscillatoriales of Cyanobacteria, their relative abundance reached the highest in algal crusts and gradually decreased with the succession of BSCs. In the bare sand, IC type autotrophic microbes were dominated by Solirubrobacterales, Hyphomicrobiales, Nitrosomonadales and Rhodospirillales were dominated in BSCs. Naviculales of Bacillariophyta dominated the ID type microbes, and their abundance reached the highest in bare sand. The community structure of IAB and IC type autotrophic microbes were similar in algal crusts and lichen crusts, but significantly different from that in bare sand and moss crusts, which also had significant differences. The variations of biological groups and soil physicochemical properties provided different microhabitats (niches) for autotrophs. The total organic carbon, total nitrogen, ammonium nitrogen, total phosphorus, available phosphorus, and pH regulated autotrophic microbial communities comprehensively. Through the screening of carbon-fixing microbes, the community composition and structure were finally changed. The study provides new evidences in understanding microbial mechanisms of carbon sequestration of BSCs in desert ecosystems.