In order to differentiate two species of Radix Puerariae (Radix Puerariae lobatae and Radix Puerariae thomsonii) and to determine major isoflavonoids (puerarin, daidzin, daidzein and genistein) in the samples, a simple high performance liquid chromatography (HPLC) method with isocratic elution employing cyclodextrins (CDs) as mobile phase additives was developed. Various factors affecting the retention of isoflavonoids in the C(18) reversed-phase column, such as the nature of CDs, the concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD) and the methanol percentage in the mobile phase, were studied. Experimental results confirmed that HP-β-CD, as a very effective mobile phase additive, could markedly reduce the retention of isoflavonoids, especially daidzein and genistein. The elution of four isoflavonoids could be achieved on a Kromasil(®) C(18) column within 56 min by using the methanol-water contained 5 mM HP-β-CD (25/75, v/v) mixture as the mobile phase. The formation of the inclusion complexes between isoflavonoids and HP-β-CD explained the modification of the retention of analytes. The apparent formation constants determined by HPLC confirmed that the stoichiometry of HP-β-CD-isoflavonoid complexes was 1:1, and the stability of the complexes depended on the size and property of isoflavonoids. The optimized method was successfully applied for the simultaneous determination of major isoflavonoids in P. lobatae and P. thomsonii samples. This work provides a useful method for the analysis of traditional Chinese herbs.