The enzymes of STARCH SYNTHASE (SS) are key players in starch biosynthesis which have proven their impact on different aspects of the starch biosynthesis and functionalities. These SSs seem to be key differences among species and organs regarding their contributions to starch synthesis. The function of StSS5 in starch synthesis of tubers was proposed, but it remains to be investigated. Here, we generated two heterozygous biallelic mutants within the 5'-untranslated region of StSS5, which resulted in the knockdown of StSS5 in a dihaploid potato AC142 through the CRISPR/Cas9 system. We investigated the role of StSS5 in potato, which has a distinct pattern of granule initiation between leaf chloroplasts and tuber amyloplasts. The ss5 mutants produce fewer starch granules in chloroplasts and attenuate starch degradation during night periods in leaves. By contrast, the ss5 mutations induce supernumerary starch granule initiations, leading to the formation of compound starch granules in amyloplasts and a relatively higher amounts of small starch granules in tubers. Additionally, these distinct starch granule initiations in the ss5 mutants were associated with a decrease in tuber fresh weight and starch accumulation. Overall, our work demonstrates that StSS5 is critical for granule initiation in tuber amyloplasts and leaf chloroplasts of potato to modulate tuber development and starch accumulation, which may provide a strategy to improve tuber starch yield and modify starch properties of potato.