Antibiotic resistant bacteria are widely spread in environments. However, the compositions of bacteria with antibiotic resistance in subtropical fish pond sediment are still unclear. In present study, to analyze the compositions of norfloxacin resistant bacteria in subtropical fish pond sediments, we selectively cultured the sediment bacteria collected from six subtropical fish ponds located in southern China using the basic medium with 50 µg/ml of norfloxacin, and analyzed their compositions using MiSeq high-throughput sequencing of 16S rRNA gene amplicons. Our results showed that various norfloxacin resistant bacteria existed in the fish pond sediments. And most of them were Gram-negative bacteria. Their spatial distribution was mainly influenced by environmental heterogeneity of the ponds. These results implied that norfloxacin resistance was widespread in Gram-negative bacteria in the fish pond sediments located in southern China. These finding provided reference information to prevent fish infected diseases and to assess the risk of antibiotic resistant bacteria in the fish pond sediments.