Phaffia rhodozyma cells were treated with the mutagenic agent NTG several times and plated on yeast-malt agar containing β-ionone as a selective medium. This mutagenesis of the yeast yielded a mutant (NCHU-FS501) with a total carotenoid content of 1454 μg g−1 dry biomass. Temperature and pH had only a slight effect on the volumetric pigment production by the red yeast, however astaxanthin yield and specific growth rate were influenced more significantly by temperature and pH. The optimum inoculum size, temperature and air flow rate for astaxanthin formation by the mutant in a bench-top fermentor were 7.5% (v/v), 22.5°C and 3.6 vvm, respectively. Glucose (1%, w/v) as carbon source yielded the highest volumetric astaxanthin production (6.72 μg ml−1). Peptone (15.8% total nitrogen) was the best nitrogen source for astaxanthin production (6.72 μg ml−1). Pigment formation by the mutant was further improved by increasing the glucose concentration to 3.5%, where the astaxanthin concentration was 16.33 μm ml−1. At 4.5% glucose or above astaxanthin formation was inhibited. Control of the pH of the fermentation broth did not improved pigment production.