Abstract RNA viruses have high genetic diversity, allowing rapid adaptation to environmental pressures, such as disinfection. This diversity increases the likelihood of mutations influencing the viral sensitivity to disinfectants. Ethanol is widely used to control viral transmission; however, insufficient disinfection facilitates the survival of less-sensitive viruses. Further, the underlying mechanisms of ethanol-induced changes in viral sensitivity remain unclear. Here, we assessed the genetic characteristics of ethanol-adapted murine norovirus (MNV) and associated changes in viral sensitivity. Experimental ethanol-facilitated MNV adaptation and subsequent genetic characteristic evaluation of the whole genome sequence was performed. MNV was exposed to 70% ethanol for 5 s to achieve ± 3-log10 inactivation. Twelve MNV populations were identified as “less sensitive,” consisting of nine treated and three control populations. Less-sensitive MNV populations exhibited significantly higher synonymous nucleotide diversity (πS) in ORF1 (p = 0.001), which encodes the non-structural protein, than sensitive populations. Ethanol sensitivity and πS were negatively correlated in ORF1 (R = − 0.49, p = 0.003), indicating that high genetic diversity in ORF1 could be linked to reduced ethanol sensitivity. This study demonstrates an association between nucleotide diversity in specific coding regions of the MNV genome and ethanol sensitivity. These findings are vital for improving disinfection methods and anticipating emerging viruses that are more resistant to disinfectants. Key points • Several MNV populations reduced sensitivity to ethanol. • Higher synonymous diversity in ORF1 linked to reduced ethanol sensitivity. • Synonymous mutations can influence viral adaptation to ethanol.