Due to global warming and changes in precipitation patterns, many regions are prone to permanent drought. Rapeseed (Brassica napus ) is one of the main sources of edible oils worldwide, and its production and yield are affected by drought. In this study, gene expression alterations under drought stress are investigated with bioinformatics studies to examine evolutionary relations of conserved motifs structure and interactions among Calvin cycle and photorespiration pathways key genes in drought-tolerant (SLM046) and drought-sensitive (Hayola308) genotypes of rapeseed. Investigating the conservation and evolutionary relationships revealed high conservation in motifs of FBPase, PRK, GlyK and NADP-ME enzymes. The analysis of protein interactions showed the correlation between FTRC, FBPase1, PRKX1, GlyKX2 and NADP-ME4 genes. Furthermore, in rapeseed, for the GlyKX2 and NADP-ME4 genes, four microRNAs of the miR172 family and four members of the miR167 family were identified as post-transcriptional regulators, respectively. The expression of ferredoxin thioredoxin reductase, fructose-1,6-bisphosphatase genes, phosphoribulokinase, glycerate kinase and malic enzyme 4 genes in the two rapeseed genotypes were evaluated by real-time qPCR method under 72h of drought stress and methanol foliar application. As a result, the highest expression levels of FTRC, PRKX1, GlyKX2, NADP-ME4 and FBPase1 were observed in methanol foliar application on the SLM046 genotype at 24h. In contrast, in methanol foliar application on the Hayola308 genotype, the highest expression levels of FTRC, PRKX1, GlyKX2, NADP-ME4 and FBPase1 were observed 8h after the treatment. Our study illustrated that methanol foliar application enhanced plant tolerance under drought stress.