In order to study adaptation of microalgae to petroleum contamination, we have examined an environmental stress gradient by crude oil contamination in the Arroyo Minero River (AMR), Argentina. Underground crude oil has constantly leaked out since 1915 as a consequence of test drilling for possible petroleum exploitation. Numerous microalgae species proliferated in AMR upstream of the crude oil spill. In contrast, only four microalgal species were detected in the crude oil spill area. Species richness increases again downstream. Microalgae biomass in the crude oil spill area is dominated by a mesophile species, Scenedesmus sp. Effects of oil samples from AMR spill on photosynthetic performance and growth were studied using laboratory cultures of two Scenedesmus sp. strains. One strain (Se-co) was isolated from the crude oil spill area. The other strain (Se-pr) was isolated from a pristine area without petroleum contamination. Crude oil has undetectable effects on Se-co strain. In contrast crude oil rapidly destroys Se-pr strain. However, Se-pr strain can adapt to low doses of petroleum (≤ 3% v/v total hydrocarbons/water) by means of physiological acclimatization. In contrast, only rare crude oil-resistant mutants are able to grow under high levels of crude oil (≥ 10% v/v total hydrocarbons/water). These crude oil-resistant mutants have arisen through rare spontaneous mutations that occur prior to crude oil exposure. Species richness in different areas of AMR is closely connected to the kind of mechanism (genetic adaptation vs. physiological acclimatization) that allows adaptation. Resistant-mutants are enough to assure the survival of microalgal species under catastrophic crude oil spill.