Sweetpotato (Ipomoea batatas (L.) Lam.) is an important multifunctional crop with great value in food supply, industrial processing and bioenergy utilization. Crude protein content (CPC) is a core target trait for sweetpotato quality breeding. To dissect the genetic basis of CPC and identify key candidate genes, we used an F₁ population of 212 individuals. CPC was measured by near-infrared reflectance spectroscopy (NIRS) in 2020 and 2021, and QTL mapping was performed using a high-density SNP genetic linkage map. Candidate genes were explored via a genome-wide association study (GWAS), multiple-database functional annotation, and quantitative real-time PCR (qPCR) validation. The results showed that: (1) CPC in the population exhibited a continuous normal distribution with high inter-year stability, and phenotypic variation was mainly controlled by genetic factors; (2) one stable minor-effect QTL for CPC, qCPC09-1, was mapped to Chr09: 7906895–8614924 bp, explaining 5.7% of phenotypic variation; (3) GWAS detected no significant SNP loci, suggesting that CPC is regulated by multiple minor-effect genes; (4) genes within the qCPC09-1 interval were significantly enriched in three protein synthesis-related KEGG pathways: ribosome, nitrogen metabolism and ubiquinone and other terpenoid–quinone biosynthesis; (5) qPCR verified that itf09g13420 and itf09g13230 were upregulated in the low-CPC parent Yushu 10 and negatively correlated with CPC, while itf09g13550 was upregulated in the high-CPC parent Xin 24 and positively correlated with CPC. These three genes exhibited expression patterns highly consistent with phenotypic differences. This study provides a theoretical basis and technical support for molecular marker-assisted breeding and elite germplasm innovation in sweetpotato.