Water stress caused by drought affects the productivity of the sugarcane crop. A breeding alternative is the selection of drought-tolerant sugarcane cultivars. The objective of this study was the in vitro screening of cultivars tolerant to water stress using polyethylene glycol (PEG) as a stressing agent. Cultivars (cv) Mex 69-290, CP 72-2086, Mex 79-431 and MOTZMex 92-207 were subjected to different concentrations of PEG 6000 (0, 3, 6 and 9% w/v) using Murashige and Skoog semi-solid culture medium. At 30 days of culture, different developmental variables and dry matter (DM), total protein (TP), proline (Pr) and glycine-betaine (GB) contents were evaluated. The results showed reduced development in cv CP 72-2086, Mex 79-431 and MOTZMex 92-207 with increasing PEG concentration. The cv Mex 69-290 showed tolerance to osmotic stress of −0.45 MPa using 3% PEG. Overall, TP content decreased with increasing PEG concentration, while DM, Pr and GB contents rose with increasing PEG concentration in all evaluated cultivars. Our results suggest that cv Mex 69-290 has a slight tolerance to water stress and could be used for rainfed cultivation with low rainfall or reduced irrigation for better water use efficiency. In conclusion, the in vitro screening technique of cultivars tolerant to PEG-induced water stress is an alternative for early determination of drought stress in sugarcane.