To satisfy future demands, the increase of wheat (Triticum aestivum L.) yield is inevitable. Simultaneously, maintaining high crop productivity and efficient use of nutrients, especially nitrogen use efficiency (NUE), are essential for sustainable agriculture. NUE and its components are inherently complex and highly influenced by environmental factors, nitrogen management practices and genotypic variation. Therefore, a better understanding of their genetic basis and regulation is fundamental. To investigate NUE-related traits and their genetic and environmental regulation, field trials were evaluated in a Central European wheat collection of 93 cultivars at two nitrogen input levels across three seasons. This elite germplasm collection was genotyped on DArTseq® genotypic platform to identify loci affecting N-related complex agronomic traits. To conduct robust genome-wide association mapping, the genetic diversity, population structure and linkage disequilibrium were examined. Population structure was investigated by various methods and two subpopulations were identified. Their separation is based on the breeding history of the cultivars, while analysis of linkage disequilibrium suggested that selective pressures had acted on genomic regions bearing loci with remarkable agronomic importance. Besides NUE, genetic basis for variation in agronomic traits indirectly affecting NUE and its components, moreover genetic loci underlying response to nitrogen fertilisation were also determined. Altogether, 183 marker-trait associations (MTA) were identified spreading over almost the entire genome. We found that most of the MTAs were environmental-dependent. The present study identified several associated markers in those genomic regions where previous reports had found genes or quantitative trait loci influencing the same traits, while most of the MTAs revealed new genomic regions. Our data provides an overview of the allele composition of bread wheat varieties anchored to DArTseq® markers, which will facilitate the understanding of the genetic basis of NUE and agronomically important traits.