In this study, Roche 454 high throughput technology was used to conduct transcriptome sequencing and establish ESTs database of the largemouth bass northern subspecies and Florida subspecies. The results showed that a total of 468 671 and 332 322 ESTs were generated from northern subspecies and Florida subspecies. The average length was 306.5 bp and 304.4 bp, respectively. Assembly of the largemouth bass northern subspecies and Florida subspecies’ high quality ESTs resulted in 42 056 and 35 743 contigs. The average length was 612.6 bp and 588.2 bp, respectively. EST database of largemouth bass northern subspecies and Florida subspecies was merged and then compared with known protein databases. 78 938 EST sequences in total were annotated. By exploring the merged largemouth bass EST library, over 25 469 and 8 547 putative SSRs and SNPs were identified. Comparison of the northern subspecies and Florida subspecies sequence was made by randomly selecting 75 contigs from the EST database. Results showed that EST sequences of the two subspecies homology were 99.2%. 