The unique bread-making properties of wheat are due to the characteristics of the grain storage proteins being able to form the gluten network. The gluten proteins are able to support the dough and bread structure and a more complex network is contributing to dough strength. The bread-making industries in many countries are preferring a relatively strong gluten for successful processing. Not only the protein content, but also the protein composition contributes to generate desired characteristics to the dough. It is well known that both genotype and growing conditions affect the grain protein composition, most of the characteristics are formed during the grain development. The aim of this work was to contribute to an understanding of how genotype and nitrogen fertilization influence the content and composition of the gluten proteins during grain development and the resulting protein characteristics at maturity, together with effects on the gluten strength. Three genotypes (two commercial varieties, one French and one Uruguayan, and one experimental line) were selected to cover a wide range of gluten strength: Alveograph W average (AW; generally defined as baking strength) in the experiment were 311, 261 and 159 J/10000, respectively, for the three varieties, thereby representing a strong, a medium and a weak genotype. The varieties were grown with three different nitrogen treatments (NT) in 3-reps block randomized trials. Grains were collected at five occasions during grain development, i.e. at Zadoks decimal codes 71, 75, 77, 85 and 92 (the latest one correlating to harvest time). Gluten properties were characterized by SE-HPLC. Mature grains samples were used to determine grain protein content (GP) and to extract flour to obtain Alveograph data. The results showed that the ratio of soluble polymeric proteins over total proteins (%PPs) obtained through SE-HPLC correlated with AW. Gluten strength of the varieties correlated positively with an increase of AW with GP. Similarly, each genotype generated a differential response of %PPs vs total proteins, sorting the genotypes in the same order as AW to GP. Furthermore, a positive correlation was found between AW and %PPs, independently of genotype and NT. Protein composition during grain filling measured by SE-HPLC indicated a similar evolution for all varieties. However, %PPs started to differentiate among NT at Zadoks 85 for all genotypes, resulting in a higher differentiation correlating with strength of the varieties. A clear relationship between protein components and dough strength was observed, regardless of genotype or NT.