Biofuel made from agricultural products has the potential in contribute to a stable supply of fuel for growing energy demands. Some salient plant traits, such as stem diameter and water content, and their relationship to other important biomass-related traits are so far poorly understood. Here, we performed QTL mapping for three stem diameter and two water content traits in a S. bicolor BTx623 x IS3620c recombinant inbred line population of 399 genotypes, and validated the genomic regions identified using genome-wide association studies (GWAS) in a diversity panel of 354 accessions. The discovery of both co-localized and non-overlapping loci affecting stem diameter traits suggests that stem widths at different heights share some common genetic control, but also have some distinct genetic influences. Co-localizations of stem diameter and water content traits with other biomass traits including plant height, flowering time and the ‘dry’ trait, suggest that their inheritance may be linked functionally (pleiotropy) or physically (linkage disequilibrium). Water content QTL in homeologous regions resulting from an ancient duplication event may have been retained and continue to have related functions for an estimated 96 million years. Integration of QTL and GWAS data advanced knowledge of the genetic basis of stem diameter and water content components in sorghum, which may lead to tools and strategies for either enhancing or suppressing these traits, supporting advances toward improved quality of plant-based biomass for biofuel production.