The rising demand for plant-based proteins has intensified interest in pulse crops due to their high protein concentration. Few studies have evaluated protein and amino acid composition or variability in cultivated lentil (Lens culinaris Medik.). We evaluated protein and amino acid composition using near-infrared reflectance spectroscopy in a diversity panel grown in four site-years in Saskatchewan, Canada, followed by genome-wide association analyses with phenology-related traits as covariates. We found little correlation between protein concentration and days from sowing to flowering, region of origin, cotyledon color, or seed size. Reproductive period was correlated with protein concentration, however. We also observed variability among environments and more variability within market classes than among them. We demonstrate the potential for breeders to identify adapted germplasm and select for increased protein and amino acid concentration and quality. We were able to identify several molecular markers for use in marker-assisted breeding to select for protein concentration or quality.