Mitochondrial reactive oxygen species (ROS) production, specifically at complex I (Cx I), has been widely suggested to be one of the determinants of species longevity. The present study follows a comparative approach to analyse complex I in heart tissue from 8 mammalian species with a longevity ranging from 3.5 to 46 years. Gene expression and protein content of selected Cx I subunits were analysed using droplet digital PCR (ddPCR) and western blot, respectively. Our results demonstrate: 1) the existence of species-specific differences in gene expression and protein content of Cx I in relation to longevity; 2) the achievement of a longevity phenotype is associated with low protein abundance of subunits NDUFV2 and NDUFS4 from the matrix hydrophilic domain of Cx I; and 3) long-lived mammals show also lower levels of VDAC (voltage-dependent anion channel) amount. These differences could be associated with the lower mitochondrial ROS production and slower aging rate of long-lived animals and, unexpectedly, with a low content of the mitochondrial permeability transition pore in these species.