Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 μg/ml dimethachlon with the resistance factor ≈10 (resistance factor was estimated as ratios between the EC50 values of resistant isolates and the average EC50 values of sensitive ones) compared to the sensitive isolates (EC50 is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum, together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.