qSsnps-5D，derived from the backbone parental line Jing 411（J411），is a major stable QTL for sterile spikelet number per spike（SSNPS）. This study aimed to decipher the genetic effect of this QTL and its use in wheat varieties. Two populations，including a recombinant inbred lines derived from the cross of Kenong 9204（KN9204）and Jing 411（KJ-RIL），and a natural mapping population comprised by 314 wheat authorized varieties or advanced lines，were involved. The experiment results showed that qSsnps-5D was confirmed in an interval of 3.41 Mb from 0.72 Mb to 4.13 Mb on chromosome 5D among 8 datasets. Excellent allele of qSsnps5D from J411 could increase spikes per plant but significantly decrease thousand kernel weight in both KJ-RIL and the natural mapping populations. However，the genetic effects of qSsnps-5D on kernel number per spike and yield per plant were differing between the two populations. Two close linkage markers of qSsnps-5D，i.e.，AX110565536 and AX-86170796 were used to specify the breeding utilization characteristics of qSsnps-5D in the natural mapping population. The excellent haplotype for qSsnps-5D（Hap-GG-CC）was often found（80.60%） in the foreign varieties. Among the Chinese varieties，higher rates of excellent haplotype（Hap-GG-CC）were found from varieties of Qinghai province，Sichuan province and Henan province，but the rates of haplotype HapGG-CC were lower in varieties of Shandong，Beijing，Shaanxi and Hebei. The rates of Hap-GG-CC in varieties released from 1980 s to present were constantly declined. To facilitate the application of qSsnps-5D in future molecular breeding programs，we developed a PCR-based InDel molecular marker named as 5D-1620921. Altogether，this study provided insights for the application of qSsnps-5D in molecular breeding programs of wheat.