A total of 4507 SSRs distributed in 4039 transcript sequences were identified from 73515 unigenes in Gossypium hirsutum shoot tip transcriptome. The frequency of unigene sequences containing SSR was 5.5% and the number of SSRs was significantly higher in non-coding regions than that in coding regions (2853/1654). The SSR repeat motifs dominated by trinucleotide repeats (51.03%), among which AAG/CTT (20.08%) accounted for the highest proportion. Dinucleotide repeats motifs (28.76%) were the next, and AG/CT (55.47%) were the dominant type. Using primers batch design software, 1569 pairs of genic-SSR primers were developed. Preliminary evaluation was conducted on four Gossypium species, TM-1 (Gossypium hirsutum L.), 3-79 (G.barbadense L.), G. herbaceum L var. africanum and G. raimondii Ulbrich.. A total of 1117 primer pairs amplified stable bands and the amplification rate is 71.2%. Six hundred and fifty pairs of primers were randomly selected and further screened in 13 accessions of five cotton species (plus G. arboreum L.). Eighty-three of these primers could amplify special bands, the PIC value ranged from 0.121 to 0.648, and the average PIC value was 0.422. Eighty pairs of primers displayed polymorphisms within G. hirsutum, and the average PIC value was 0.336. Five among the 83 genic-SSR primer pairs which distinctly revealed polymorphism between the laboratory mapping parents of Lumianyan22 and Luyuan343 were selected and carried on linkage analysis, four of the SSRs were successfully integrated into intraspecific genetic linkage map previously constructed in our lab. The results enrich the quantity of cotton genus genic-SSR for mapping and association analysis and related research on cotton species.