Temperature, which is an important environmental factor in broiler farming, can significantly influence the deposition of fatty acids in muscle. 300 one-day-old broiler chicks were randomly divided into three groups and reared at high, medium and low temperatures (HJ, MJ and LJ), respectively. Breast muscle and jejunal chyme samples were collected and subjected to analyses of fatty acid composition and 16S rRNA gene sequencing. Through spearman's rank correlation coefficient, the data were used to characterize the correlation between jejunal microbial diversity and muscle fatty acid deposition in the broilers. The results showed that Achromobacter, Stenotrophomonas, Pandoraea, Brevundimonas, Petrobacter and Variovorax were significantly enriched in the MJ group, and all of them were positively correlated with the fatty acid profiling of muscle and multiple lipid metabolism signaling pathways. Lactobacillus was significantly enriched in the HJ group and exhibited a positive correlation with fatty acid deposition. Pyramidobacter, Dialister, Bacteroides and Selenomonas were significantly enriched in the LJ group and displayed negative correlation with fatty acid deposition. Taken together, this study demonstrated that the jejunal microflora manifested considerable changes at high and low ambient temperatures and that jejunal microbiota changes were correlated with fatty acid deposition of muscle in broilers.