Improving total protein content that has a higher concentration of essential amino acids and a more balanced composition will enhance the health benefit potential of rice grain. Rice is a good source of B vitamins and minerals. It has been known also to provide advantageous phytochemicals which are known to link to lipid lowering, antioxidant, anti-inflammatory, glucoregulatory, and anti cancer agents. The objective of this research was to determine amino acid profiles and molecular characteristics of five rice lines possessing improved grain protein content. Three newly developed high protein rice lines were derived from the rice cultivar Francis, designated as FRN936, FRN937, and FRN783, with protein content improvement ranging from 10 to 12.5% (w/w: protein/grain weight) from their original protein content of 8%. These lines are fertile with grain and other phenotypic characteristics that are similar to Francis. Two additional high protein lines, WLS07 and WLS97, were developed from the cultivar Wells. Amino acid profiles from each of five high protein lines developed varied. The composition of essential amino acids in the FRN936 line, for example, is 18, 19, 35, 13, 40, 44, 15, 37, and 30% more in lysine, threonine, phenylalanine, methionine, isoleucine, valine, tryptophan, histidine, and arginine, respectively, compared with that found in its parental Francis wildtype check. While WLS97 has 47, 11, 46, 18, 37, 34, 30, 20, and 21% more in lysine, threonine, phenylalanine, methionine, isoleucine, valine, tryptophan, histidine, and arginine, respectively, compared with that found in its parental Wells. Improved total protein content with more essential amino acids is an important step to the enhancement of rice health benefit potential. Genes coding for key enzymes to essential amino acid synthesis, such as DHDPS, were used to characterize high protein lines. Molecular characteristics of these lines will be discussed.