Abstract: In order to study the spatial distribution of microorganisms and volatile compounds in pit mud, solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to qualitatively and quantitatively analyze the main volatile compounds in pit mud, and Illumina MiSeq sequencing was used to analyze the composition of microbial community in pit mud. The results of the study showed that the volatile components in the pit mud were mainly esters and acids, accounting for 73.01% to 98.36%, and they continued to increase with the increase of the depth of the pit. A total of 10 phyla, 15 classes, 21 orders, 42 families, and 64 genera of prokaryotic microorganisms were detected in the pit mud samples. The dominant flora accounted for 98.86%~99.93% of the total number of bacteria at the phylum level, and Firmicutes was the absolute dominant flora. The Shannon index increased from 2.130 to 4.970, the Simpson index increased from 0.557 to 0.932, and the ACE index and Chao1 index were the highest at the bottom of the pool, at 114.523 and 114.50, respectively. Results of α-diversity indicated that the abundance and diversity of microbial flora in the lower part of the pool wall and pit mud at the bottom of the pool were higher than that in the upper and middle parts of the pool wall, which was consistent with the type and content distribution of volatile substances. Dominant bacteria such as Clostridium and Methanobacter were the main microorganisms which could carry out microbial metabolism and inter-species material exchange, and produce important aroma substances and their precursors. As the depth of space increased, they continued to react and accumulate, closely related to spatial location of the mud. Spatial difference was an important reason for the differences in the composition of prokaryotic microbes and volatile substances in different spatial locations of pit mud.