The yellowfin seabream (Acanthopagrus latus) is an economically important commercial mariculture fish in China and Southeast Asia. Only a few simple sequence repeats (SSRs) of A. latus have been isolated and reported, which has hindered breeding progress. A total of 318,862 SSRs were isolated and characterized from the A. latus genome in this study. All SSRs were 9,069,670 bp in length, accounting for 1.32% of the genome. The density and frequency of SSRs were 468.40 loci/Mb and 13,323.19 bp/Mb, respectively. The major SSRs were dinucleotides (accounting for 76.92%), followed by trinucleotides (15.75%). The most abundant SSR motif was (AC)n (168,390, accounting for 53%), with the highest frequency (245.78 loci/Mb) and density (7304.18 bp/Mb). Most SSRs were located in non-coding regions, such as intergenic regions (34.54%) and introns (56.91%). SSR-containing exons were distributed into 51 gene ontology (GO) terms and significantly enriched in immunity- and growth-related pathways. A total of 217,791 SSR markers were successfully designed. Nine SSR markers were amplified in 29 A. latus individuals, and eight of them possess high polymorphism. The cross-species transferability of 33 out of the 37 tested loci were successfully amplified in Acanthopagrus schlegelii. These results lay the foundation for the molecular marker-assisted breeding and genetic information assessment of A. latus.