Cultivated lentil (Lens culinaris Medik.) has a relatively narrow genetic base which poses many challenges to the improvement of the crop. Introgressing traits like biotic and abiotic stress resistance from wild relatives is often hindered by linkage drag of undesirable traits. Lentil is a nutritious staple crop, rich in protein, complex carbohydrates, vitamins, and minerals. This study aimed to investigate the effects of crossing with wild lentil on seed protein and amino acid contents. An interspecific recombinant inbred line (RIL) population, LR-68, generated from a cross between Lens culinaris and Lens orientalis was grown in four site-years in Saskatchewan, Canada. LR-68 was evaluated for protein and 18 amino acid contents using near-infrared (NIR) spectroscopy. Quantitative trait loci (QTL) analyses were then conducted to identify regions of the genome associated with protein and amino acid contents. LR-68 exhibited significant variation for protein and amino acid content, with protein content ranging from 24.9 to 34.9% on a dry basis. Protein and amino acid contents were highly correlated with each other. The correlations between protein content and days to flower, days to maturity, seed size, and seed shape were minor. Protein and amino acid contents were significantly affected by genotype, environment, and genotype by environment interaction. Genetic analyses revealed three QTL dense regions, with QTL for protein and multiple amino acids present at the same loci. At two of the three QTL dense regions, genotypes containing the allele from the wild parent, L. orientalis, had significantly higher protein and amino acid contents. Selecting for increased protein content when crossing with L. orientalis will also increase individual amino acid contents.