Yellow betaxanthins and red purple betacyanins are genetically and structurally closely related pigments, hence making their separation from each other difficult and tedious. Fractionation of betalains into betacyanin and betaxanthin could be achieved by employing aqueous two-phase extraction (ATPE). Prior to fractionation, purification of betalains (devoid of sugars) was achieved employing ATPE comprising PEG 6000/ammonium sulphate. At a high tie line length (TLL) of 50.65%, phase volume ratio 1.96 and pH around 5, maximum differential partitioning was observed, wherein 92.62% of the betalains partitioned to the PEG-rich (top) phase while 72.38% of sugars partitioned to the salt-rich (bottom) phase. Fractionation of betalains into betacyanin and betaxanthin was achieved by multistage ATPE. At the end of the third stage ATPE, the bottom phase had only betaxanthin without any residual betacyanin. The fractions of betacyanin and betaxanthin were analyzed by spectrophotometric method and confirmed by HPLC. (C) 2014 Elsevier Ltd. All rights reserved.