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SSR molecular markers were obtained based on abundant transcriptome data under different waterlogging conditions in order to develop appropriate biological tools and explore the molecular mechanism of hot pepper response to waterlogging stress in this experiment. The results were as follows: (1) A total of 128 939 Unigenes were obtained from transcriptome analysis of hot pepper. The total length, average length, and GC content were 55 082 725 bp, 1 101 bp and 40.57%, respectively. Compared with seven major functional databases, 102 123 (NR, 79.20%), 110 157 (NT, 85.43%), 70 203 (SwissProt, 54.45%), 73 539 (KOG, 57.03%), 77 646 (KEGG, 60.22%), 77 442 (GO, 60.06%) and 68 216 (Pfam, 52.91%) Unigenes obtained functional annotations. It was found that lipid metabolism, carbohydrate metabolism, amino acid metabolism, environmental adaptation, secondary metabolite biosynthesis, signal transduction and translation played important roles in the response to waterlogging stress. (2) A total of 26 574 SSR loci were found in 24 889 Unigenes from hot pepper transcriptome data. The frequency of SSR was 20.61%, of which single nucleotide repeat accounted for the highest ratio (37.26%), followed by tri-nucleotide (31.00%) and di-nucleotide (25.44%) repeat types, accounting for 93.70% of the total EST-SSR. The motifs A/T, AG/CT and TC/GA, followed by AT and TA, were the most abundant in mono-nucleotide and di-nucleotide. The most common types of tri-nucleotides were TTG/CAA and ACA/TGT. (3) 10 002 pairs of EST-SSR primers were designed with Primer 3. A total of 30 pairs of primers were randomly selected for amplification test, and all of them were effective. Seven pairs of primers could amplify the target bands in three hot pepper germplasms. To sum up, the motif structure of the most dominant SSR repeat type in hot pepper is basically similar to that of other varieties, and the molecular mechanism of hot pepper waterlogging stress response is preliminarily explored, and its EST-SSR markers are developed to provide reference for hot pepper waterlogging-tolerant genetic breeding.