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Astaxanthin has recently attracted considerable attention for its biological properties such as the antioxidant activity as well as a coloring agent used for farmed fish. However, its biological production, mainly by a green microalgae Haematococcus pluvialis , is costly because of its fastidious growth characters. Therefore, for future applications of biological astaxanthin production in aquaculture, non-fastidious microalgal strains were isolated from environmental samples, examined for their astaxanthin-accumulating activity, and characterized phylogenetically. While β-carotene was found in all tested isolates, one isolate, GK12, from activated sludge of a sewage treatment plant accumulated 2.5 ± 0.36 mg/g dry cell of free astaxanthin de novo under photoautotrophic culture condition, which was comparable to photoautotrophic cultures of other known astaxanthin-accumulating microorganisms. Mixotrophic culture conditions increased GK12 biomass, but astaxanthin content was decreased, suggesting that the simple photoautotrophic cultivation is more efficient way for GK12 to produce astaxanthin than mixotrophic cultivation. A phylogenetic study of SSU rDNA strongly suggested that GK12 is a novel species in the genus Monoraphidium , Chlorophyta. Besides making efforts to increase astaxanthin-productivity of known astaxanthin accumulators, it is also beneficial to study GK12 from the view point of applications to aquaculture.