This study investigates a lethal mitonuclear incompatibility in naturally hybridizing swordtail fish species, Xiphophorus birchmanni and Xiphophorus malinche. The incompatibility involves three genes that form part of respiratory complex I, leading to reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, while heterozygous individuals show reduced complex I function. The effects of different genetic interactions on survival are non-additive, highlighting the complexity of hybrid incompatibilities. The evolutionary history of the genes involved shows signals of accelerated evolution and evidence of introgression between species via hybridization. This study provides insights into the genetic and evolutionary forces driving the emergence of hybrid incompatibilities and suggests that protein complexes may be critical sites of hybrid breakdown.This study investigates a lethal mitonuclear incompatibility in naturally hybridizing swordtail fish species, Xiphophorus birchmanni and Xiphophorus malinche. The incompatibility involves three genes that form part of respiratory complex I, leading to reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, while heterozygous individuals show reduced complex I function. The effects of different genetic interactions on survival are non-additive, highlighting the complexity of hybrid incompatibilities. The evolutionary history of the genes involved shows signals of accelerated evolution and evidence of introgression between species via hybridization. This study provides insights into the genetic and evolutionary forces driving the emergence of hybrid incompatibilities and suggests that protein complexes may be critical sites of hybrid breakdown.