The pyrethroid deltamethrin (DTM) is used to treat Atlantic salmon (Salmo salar) against salmon louse (Lepeophtheirus salmonis) infestations. However, DTM resistance has evolved in L. salmonis and is currently common in the North Atlantic. This study aimed to re-assess the association between DTM resistance and mitochondrial (mtDNA) mutations demonstrated in previous reports. Among 218 L. salmonis collected in Scotland in 2018-2019, 89.4% showed DTM resistance in bioassays, while 93.6% expressed at least one of four mtDNA single nucleotide polymorphisms (SNPs) previously shown to be resistance associated. Genotyping at further 14 SNP loci allowed to define three resistance-associated mtDNA haplotypes, named 2, 3 and 4, occurring in 72.0%, 14.2% and 7.3% of samples, respectively. L. salmonis strains IoA-02 (haplotype 2) and IoA-10 (haplotype 3) both showed high levels (~ 100-fold) of DTM resistance, which was inherited maternally in crossing experiments. MtDNA haplotypes 2 and 3 differed in genotype for 17 of 18 studied SNPs, but shared one mutation that causes an amino acid change (Leu107Ser) in the cytochrome c oxidase subunit 1 (COX1) and was present in all DTM resistant while lacking in all susceptible parasites. We conclude that Leu107Ser (COX1) is a main genetic determinant of DTM resistance in L. salmonis.