Hypoxia is a pervasive stressor in aquatic environments, and both phenotypic plasticity and evolutionary adaptation could shape the ability to cope with hypoxia. We investigated evolved variation in hypoxia tolerance and the hypoxia acclimation response across fundulid killifishes that naturally experience different patterns of hypoxia exposure. We compared resting O2 consumption rate (MO2), and various indices of hypoxia tolerance (critical O2 tension [Pcrit], regulation index [RI], O2 tension [PO2] at loss of equilibrium [PLOE], and time to LOE [tLOE] at 0.6 kPa O2) in Fundulus confluentus, F. diaphanus, F. heteroclitus, F. rathbuni, Lucania goodei, and L. parva. We examined the effects of chronic (28 d) exposure to constant hypoxia (2 kPa) or nocturnal intermittent hypoxia (12 h normoxia: 12 h hypoxia) in a subset of species. Some species exhibited a two-breakpoint model in MO2 caused by early, modest declines in MO2 in moderate hypoxia. We found that hypoxia tolerance varied appreciably across species: F. confluentus was the most tolerant (lowest PLOE and Pcrit, longest tLOE), whereas F. rathbuni and F. diaphanus were the least tolerant. However, there was not a consistent pattern of interspecific variation for different indices of hypoxia tolerance, with or without taking phylogenetic relatedness into account, likely because these different indices are underlaid by partially distinct mechanisms. Hypoxia acclimation generally improved hypoxia tolerance, but the magnitude of plasticity and responsiveness to different hypoxia patterns varied interspecifically. Our results therefore suggest that hypoxia tolerance is a complex trait that is best appreciated by considering multiple indices of tolerance.