Due to the intensification of rice cultivation in Spain and the repeated use of herbicides as an effective tool to control weeds, one important fact is the appearance of resistant populations of weeds, which are no longer controlled effectively at field doses. Therefore, it is necessary to increase doses to such an extent as to produce phytotoxicity in the rice crop. In paddy fields in Badajoz (Spain), a few years ago, populations of Cyperus difformis resistant to bensulfuron-methyl appeared. In order to characterize this resistance, whole plant assays were carried out in fourteen populations, eight of them originating from treated Spanish paddy fields. The results were expressed as resistance factor (RF) = ED50(R)/ED50(S). Two populations of Cyperus difformis, resistant (R) and tolerant (T) to bensulfuron-methyl, displayed resistance factors greater than 30 and 12.5, respectively. Furthermore, in order to evaluate the existence of a possible cross-resistance, the recommended field doses of 3 other herbicides (imazamox, ethoxysulfuron and bentazone + MCPA) were applied to the plants. All populations were susceptible to both imazamox and bentazone + MCPA and two populations showed cross-resistance to ethoxysulfuron. ALS activity was assayed in vitro in four biotypes, the R/S ratios of the I50 values calculated indicate that resistance involves a reduction in the sensitivity of the ALS mainly in the R population (biotype 4) to bensulfuron-methyl and ethoxysulfuron, displaying R/S ratios of 37500 and 142857, respectively, which may be attributed to inherent differences in the ALS protein itself and/or in the stability of the enzyme.