Temperature stress in invertebrates is known to be reflected by changes in catecholamine levels. However, the mechanisms of stress response are not fully understood. Groundwater and surface water amphipods are expected to be differently adapted to temperature elevations due to the different temperature regimes in their habitats and consequently, show a different stress response. No data have been published so far regarding the effects of stress on catecholamine patterns in groundwater invertebrates and accordingly, comparisons with surface water fauna are also missing. In this study, we compared the average catecholamine levels in two taxonomically related amphipod species: Niphargus inopinatus, living in groundwater with constant water temperatures throughout the year, and Gammarus pulex, a surface water stream amphipod frequently exposed to diurnal and seasonal temperature fluctuations. Furthermore, we tracked the immediate changes in whole-animal catecholamine levels in response to heat stress in both species. Pronounced differences in the catecholamine levels of the two species became apparent, with the average dopamine (DA) level of N. inopinatus being almost 1000times higher than that in G. pulex. The noradrenaline (NA) concentrations in N. inopinatus were on average two orders of magnitude higher than in G. pulex, and for adrenaline (A), the difference constituted one order of magnitude. When exposed to short-term heat stress, both species showed a response in terms of catecholamine levels, but the observed patterns were different. In N. inopinatus, temperature stress was reflected by the appearance of adrenaline, while in G. pulex a significant increase in noradrenaline levels occurred in the treatment with the highest temperature elevation.