Stem strength, as an estimate of forage intake potential, could be a useful selection parameter in alfalfa (Medicago sativa L.) breeding programs, but there is little information on sampling techniques or the effect of tissue weakening by bacterial or fungal incubation. This study investigated the effect of stem position (top, middle, bottom) and incubation with a water control, Ruminococcus flavefaciens or Neocallimastix MC-2 on stem shear strength of four alfalfa genotypes, Stem diameter was closely associated with shear force in all treatments and was used as a covariate in statistical analyses. Shear strength increased from the top to the bottom of all genotypes regardless of incubation. Both bacterial and fungal incubation reduced (P<0.01) shear strength, with the fungus reducing shear strength to the greatest extent, Genotype differences (P<0.05) across incubation treatments were detected in the bottom stem portions only, There were no genotype X incubation treatment interactions within any stem position, but genotype X position effects (P<0.01) were detected in the water incubation control, The bottom sections of alfalfa showed the largest differences and would provide the best sample material to estimate forage intake potential. Stem diameter should be used as a covariate.