The top-gray chalkiness of hybrid rice (Oryza sativaL.) seeds is a typical phenomenon in hybrid rice seeds. The chalky part of the grain is infected and is the inoculum to infect the normal seeds during storage and soaking. These seed-associated microorganisms were cultivated and sequenced using metagenomics shotgun sequencing to obtain more comprehensive information on the seed-associated microorganisms in this experiment. The results showed that fungi could grow well on the rice flour medium, similar to the ingredients of rice seed endosperms. After the assembly of metagenomic data, a gene catalog was established, comprising 250,918 genes. Function analysis showed that glycoside hydrolases were the dominant enzymes, and the genusRhizopusaccounted for the dominant microorganisms. The fungal speciesR. microspores,R. delemar, andR. oryzaewere likely to be the candidate pathogens in the top-gray chalky grains of hybrid rice seeds. These results will provide a reference for improving hybrid rice processing after harvest.