Interspecific hybridization between commercial and wild canes followed by backcrossing may transfer favorable alleles responsible for drought tolerance in sugarcane. Our study aimed to assess the distribution of BC₁ individuals on leaf anatomy and to classify them regarding heterosis values. Five BC₁ populations were established using a commercial Saccharum spp. hybrid as a donor female and the F₁ interspecific hybrids as recurrent males. Leaf anatomy included leaf thickness (LT), cuticle thickness (CT), the vertical length of bulliform cell (VBC), stomatal crypt depth (SCD), percent CT, percent VBC, and percent SCD. The anatomical traits of BC₁ showed high phenotypic variations, and all populations can be divided into three groups based on their heterosis values. Heterosis seemed to be genotype and trait dependent as the estimates varied considerably across populations and observed traits, ranging from negative on LT to positive on VBC. Group I (BC₁-1) showed positive heterosis on percent CT, percent VBC, and percent SCD. Dendrogram analysis revealed that some clones in population BC₁-1 were promising regarding stalk weight and leaf anatomy, making them desirable for further clone selections. Backcrossing with commercial canes resulted in higher BC₁ means than their mid-parents despite low heterosis on leaf anatomy.