Knowledge of genetic variations can provide clues into the molecular mechanisms regulating key crop traits. Sweet potato (Ipomoea batatas (L.) Lam.) is an important starch-producing crop, but little is known about the genetic variations in starch biosynthesis and sucrose metabolism genes. Here, we used high-throughput sequencing of pooled amplicons of target genes to identify sequence variations in 20 genes encoding key enzymes involved in starch biosynthesis and sucrose metabolism in 507 sweet potato germplasms. After filtering potential variations between gene copies within the genome, we identified 622 potential allelic single nucleotide polymorphisms (SNPs) and 85 insertions/deletions (InDels), including 50 non-synonymous SNPs (nsSNPs) and 12 frameshift InDels. Three nsSNPs were confirmed to be present in eight sweet potato varieties with various starch properties using cleaved amplified polymorphic sequence (CAPS) markers. Gene copy with loss of the fifth intron was detected in IbAGPb3 genes, and loss of multiple introns were observed in IbGBSS1-1 genes and various among germplasms based on intron length polymorphism (ILP) markers. Thus, we identified sequence variations between germplasms in 20 genes involved in starch biosynthesis and sucrose metabolism, and demonstrated the diversity in intron-loss alleles among sweet potato germplasms. These findings provide critical genetic information and useful molecular markers for revealing regulatory mechanism of starch properties.