One of the negative aspects of the intensive use of herbicides is related to the selection of resistant biotypes (Gasquez 2001). Of all biotypes resistant to herbicides, 93 species do not respond to sulfonylurea herbicides (ALS-inhibiting herbicides). The acetolactate synthase (ALS) enzyme is the first step in biosynthesis of a branched chain amino acids (valine, leucine, isoleucine). In Poland the problem of resistance to sulfonylurea herbicides has been discussed since 2001 (Rola and Marczewska 2002). Resistance tests of Apera spica-venti biotypes were conducted in the greenhouse conditions. Chlorsulfuron was applied at the four-leaf stage of development at rates ranging from 11.25 to 360 g/ha. In confirmation of resistance to chlorsulfuron as identified in biological tests, the chemical analyses were performed. The analyses investigated the influence of different doses of chlorsulfuron on free amino acids content in the aboveground part of resistant and susceptible Apera spica-venti. The analyses were carried out applying high performance liquid chromatography method (HPLC). The resistance of the biotype was confirmed in amino acids analysis. In the resistant biotype followed the increase of valine, leucine and isoleucine concentration in comparison with untreated plants and those susceptible to chlorsulfuron biotype.