Background and objectives: In temperate rice growing regions, the application of nitrogen (N) fertilizer is often split into two applications to reduce the risk of cold-induced sterility associated with high N uptake. This strategy involves a basal N application applied pre-permanent water (PW) and the second application following panicle initiation (PI). Split N application generally increases crop yield, however, data describing the impact on grain quality are relatively sparse. The objective of this study was to determine the effect of the split N method on grain quality parameters. We analyzed grain quality under various pre-PW and PI N rates. Findings: N applied pre-PW increased head rice yield (HRY; the percentage of grain that remains whole or at least 75% of the original length after milling) in all tested cultivars, however, the effect of splitting the same total N rate into two doses on HRY was cultivar specific. N rate applied pre-PW also showed a positive and negative relationship with milled grain yellowness (b*) and brightness (L*), respectively. The effect of N on amylose content was cultivar specific. The changes in quality parameters through pre-PW N application were associated with protein content. However, total protein content did not correlate as strongly with quality parameters in the split treatments compared with the single-dose treatments. Analysis of protein composition revealed N rate and timing affected protein composition, which had significant impacts on quality parameters, and the effects were cultivar specific. Conclusion: These results indicate altering the nutritional management of rice changes the protein composition affecting grain quality parameters. Significance and novelty: This study demonstrates the importance of N rate and timing of application in regard to grain quality and the significant role protein composition plays in quality characteristics.