Selenium speciation analysis is usually carried out using complex hyphenated analytical systems such as LC-ICP-MS. Here we present a novel selenium speciation approach based on a sequential derivatization and extraction combined with gas chromatography mass spectrometry for the simultaneous determination of selenite, selenate, and selenocyanate in aqueous mine wastewater samples. Selenocyanate was derivatized with triethyloxonium tetrafluoroborate to ethylselenocyanate, which was extracted into chloroform, following which the sample was split into two aliquots. One aliquot was acidified and 3,5-bis(trifluoromethyl)-o-phenylenediamine was used for the novel derivatization of selenite to 4,6-bis(trifluoromethyl)-2,1,3-benzoselenadiazole, for the determination of selenite. For the second aliquot, concentrated hydrochloric acid was added along with 4-nitro-o-phenylenediamine to simultaneously reduce selenate to selenite and derivatize the combined “selenite + selenate” fraction to 5-nitro-2,1,3-benzoselenadiazole. The benzoselenadiazoles were extracted with chloroform and all extracts were combined for GC–MS analysis. Low ng g−1 detection limits were reported for all three species. The method is unhindered by concentrations of chloride and sulphate up to 3%, as well as nitrate concentrations up to 3% for selenocyanate and selenite analysis, with minor losses in sensitivity for selenate up to 100 ppm nitrate, making the method particularly suitable for aqueous mine waste characterization. Quantitative trace selenium speciation was achieved using cost-effective materials and apparatus on a simple-to-operate benchtop instrument. The novel methodology was tested on gold mine wastewater samples; comparing to total selenium, a 63–149% recovery as the sum of species was observed. Additionally, this novel speciation approach was compared to LC-ICP-MS based selenium speciation and a reasonable agreement was found in the species distribution.