A method was developed for identification of diastereomers of 2-decarboxy-betacyanins and 2,17-bidecarboxy-betacyanins chromatographed in reversed-phase high-performance liquid chromatography (HPLC) as pairs of unknown elution order. The method was based on alkaline hydrolysis of selected betacyanin and decarboxylated betacyanin mixtures and subsequent cross-recondensation of the hydrolysates. The arising intermediate derivatives of decarboxylated betalamic acid and cyclo-dopa were stable enough for subsequent recondensation. Generated diagnostic pigments as the recondensation products were monitored by HPLC-diode-array detection-electrospray ionisation mass spectrometry. The isoforms (15R) of 2-decarboxy-betacyanins and 2,17-bidecarboxy-betacyanins were eluted earlier than the 15S forms, in contrast to betacyanins and 17-decarboxy-betacyanins, but in accordance with betaxanthins. 2,17-Bidecarboxy-betanin/2,17-bidecarboxy-isobetanin, being not resolved in reversed-phase HPLC, were partially separated by ion-pair chromatography and under these conditions their order of elution was the same as that of acylated 2,17-bidecarboxy-betacyanins. The method allows complete identification of all the decarboxylated groups of betanin, phyllocactin and hylocerenin as well as other betacyanins in biological or pharmaceutical material.