Protein content (PC) is a crucial factor that determines the end-use and nutritional quality of wheat (Triticum aestivum). Glutamine synthetase (GS), which is a major participant in nitrogen metabolism, can convert inorganic nitrogen into organic nitrogen. Although many studies have been conducted on PC and GS, a dynamic analysis of all of the filling stages has not been conducted. Therefore, 115 F9-10 recombinant inbred wheat lines of 'R131/R142' were used to analyze PC and GS activity during different developmental stages, using the conditional quantitative trait loci (QTL) mapping method. Twenty-two and six conditional QTL were detected for PC and GS activily, respectively. More QTL in leaf PC were detected during the early filling stages than in the later filling stages. Grain PC QTL displayed different dynamic variations to leaf PC QTL during the entire grain-filling stages. All of the QTL were expressed differently over time, and nine conditional QTL were detected across two filling stages. QTL with similar functions may have tended to group in specific locales. This study provides dynamic genetic information on protein accumulation during grain-filling stages.