In this study, we investigated resistance traits to the sugarcane borer Diatraea saccharalis Fab. (Lepidoptera: Crambidae) in the leaves and stalks of six sugarcane cultivars in a series of greenhouse and laboratory assays. Investigation of plant factors and infestation rates to better discriminate stalk damage by the sugarcane borer indicated that infestation of 7-month-old, single plants with 20 larvae at the third or fourth instar per plant was suitable to assess tunneling length. Three cultivars (i.e. SP803280, RB928064, and RB835486) had lower stalk damage (i.e. tunnel length) than cultivar SP891115, which exhibited relatively greater susceptibility to tunneling by the borer. The time required for the larvae to enter the sugarcane stalk was longer for cultivar SP803280, indicating resistance traits on the stalk surface, which correlated with lower stalk damage. Larvae feeding on SP813250 stalks had the lowest weight gain, indicating that this cultivar has resistance traits to larval development within its stalks. Cultivars RB867515 and SP891115 resulted in the highest mortality of early-stage larvae feeding on leaves, indicating the presence of resistance factors in their leaves. Multi-trait cluster and principal component analyses placed the cultivars into three and four clusters, respectively. The cultivars placed in different groups that exhibited resistance to leaf feeding, stalk entrance, and tunneling by the sugarcane borer could be used for crossings in sugarcane breeding programs with the goal of obtaining higher levels of resistance to D. saccharalis.