Root system architecture of plant plays a key role in water and nutrient uptake from the soil, provides anchorage and acts as a storage organ. In this current research, we have focused on the molecular and physiological basis of root system variation in canola (Brassica napus L.). Genome wide association mappings in a diverse canola germplasm panel with ~37,500 and ~30,200 single nucleotide polymorphism (SNP) markers were conducted under greenhouse and field conditions, respectively. A total of 52 significant SNP markers associated with different root architectural traits were identified in the greenhouse study. Majority of the markers were distributed on five chromosomes, A01, A02, A04, C03 and C06, of B. napus. Twenty-two candidate genes related to root growth and development were detected within 50 kbp upstream and downstream of the significant markers. Three of these candidate genes, P-glycoprotein 6 (PGP6), Tetraspanin 7 (TET7) and ARABIDILLO-2, were co-localized with three markers on chromosome C03, A01 and A04, respectively. In the field study, 31 significant SNP markers associated with different root traits were detected. A total of 15 root related candidate genes were identified within 100 kbp upstream and downstream of different significant markers. We also analyzed and compared the transcriptomes from the root systems of spring (weak root system) and winter (vigorous root system) growth habits at two different time points, 30 and 60 days. A total of 169,646 transcripts were analyzed, of which, 582 and 555 transcripts were found to be significantly differentially expressed between spring and winter types at 30 and 60 days, respectively. Several cytokinin and gibberellin associated genes and genes sets were found to be upregulated in spring type compared to winter type at 60 days. Cytokinin has proven inhibitory effect on root system architecture in different crops, whereas, gibberellin promote root elongation but inhibit lateral root growth. Therefore, we suggest that cytokinin and gibberellin may play an important role in root system variation between spring and winter growth habits. Significant marker loci, candidate genes and transcriptome profile identified in this research will assist future research to understand the root system variation in rapeseed/canola.