Zhenjiang Yao meat is a traditional variety of cooked marinated and jellied pork food in China. It is usually stored refrigerated to prevent gelatine liquefaction. Our understanding of the bacterial populations found in Zhenjiang Yao meat is limited. This study was designed to explore both the bacterial diversity and the main bacterial flora of Zhenjiang Yao meat using pyrosequencing of tagged amplicons from the V3 and V9 regions of the 16S rRNA gene. A total of 53,363 bacterial sequences from five samples and an average 10,672 reads of each sample were acquired and used in the analysis of microbial diversity. The bacterial diversity was observed to increase weekly, and the main bacterial flora changed significantly under refrigerated vacuum-packaged storage. The predominant bacteria were Vibrio during the first 7 days of storage, whereas Shewanella, Lactobacillus, Lactococcus, Yersinia, and other Enterobacteriaceae members were the main groups after 15 days in refrigerated storage. The bacterial population changed significantly after 15 days, and the bacterial communities in the 30 days sample were significantly different from all other samples. In comparison with culture-dependant method, pyrosequencing provides a more comprehensive estimate of bacterial diversity and more close to the real bacterial composition in Zhenjiang Yao meat.