Quinoa (Chenopodium quinoa Willd.) is a crop that has great potential for increased cultivation in diverse climate regions. The seed protein quality obtained from this crop is high concerning the requirements to meet human nutritional needs, but the seed protein content is relatively low if compared to crops such as grain legumes. Increased seed protein content is desirable for increasing the economic viability of this crop in order for it to be used as a protein crop. In this study, we characterized three genotypes of quinoa with different levels of seed protein content. By performing RNA sequencing of developing seeds, we determined the genotype differences in gene expression and identified genetic polymorphisms that could be associated with increased protein content. Storage nutrient analyses of seeds of three quinoa genotypes (Titicaca, Pasankalla, and Regalona) from different ecoregions grown under controlled climate conditions showed that Pasankalla had the highest protein content (20%) and the lowest starch content (46%). Our seed transcriptome analyses revealed highly differentially expressed transcripts (DETs) in Pasankalla as compared to the other genotypes. These DETs encoded functions in sugar transport, starch and protein synthesis, genes regulating embryo size, and seed transcription factors. We selected 60 genes that encode functions in the central carbon metabolism and transcription factors as potential targets for the development of high-precision markers. Genetic polymorphisms, such as single nucleotide polymorphisms (SNPs) and base insertions and deletions (InDels), were found in 19 of the 60 selected genes, which can be further evaluated for the development of genetic markers for high seed protein content in quinoa. Increased cultivation of quinoa can contribute to a more diversified agriculture and support the plant protein diet shift. The identification of quinoa genotypes with contrasting seed quality can help establish a model system that can be used for the identification of precise breeding targets to improve the seed quality of quinoa. The data presented in this study based on nutrient and transcriptome analyses contribute to an enhanced understanding of the genetic regulation of seed quality traits in quinoa and suggest high-precision candidate markers for such traits.