A relatively simple, sensitive, selective, automatic fluorimetric method for the simultaneous determination of chromium(III) and chromium(VI) by flow injection analysis (FIA) was developed. The method is based on the selective oxidation of the non-fluorescing reagent 2-(α-pyridyl)thioquinaldinamide (PTQA), which with CrVI yields an intensely fluorescent product (λex = 360 nm; λem = 500 nm). Chromium(III) is oxidized on-line to CrVI with sodium metaperiodate and the CrVI is subsequently treated with PTQA. Fluorescence due to the sum of CrIII and CrVI is measured and CrIII is determined from the difference in fluorescence values. The effects of various analytical parameters, such as acidity, flow rate, sample volume, temperature, reagent concentration and interfering species, were studied. Kinetic studies using both the stopped-flow technique and the FIA procedure were utilized in order to study and optimise the oxidation conditions for CrIII on the basis of its oxidation efficiency. The calibration graphs were rectilinear in the ranges 0.1–10 µg ml–1 for CrVI and 0.1–1.0 µg ml–1 for CrIII. The method was successfully verified by performing recovery experiments of Cr in several standard reference materials (peach leaves, sediments and tea), and it was applied to the speciation analysis of CrIII–CrVI in environmental waters (mineral, tap and distilled water), a food sample (tomato juice) and synthetic mixtures. Up to 30 samples per hour can be analyzed with a relative standard deviation of about 0.1–2%.