This study determined the inter-reader and intra-reader reliability of lower limb frontal plane alignment measures obtained from digital radiographs using a computer software program. Measurements of lower limb frontal plane alignment were obtained from over 3000 full limb digital radiographs of both limbs of persons ‘at risk’ for developing knee osteoarthritis (OA), as part of the Multicenter Osteoarthritis Study (MOST). Three trained clinicians used a computer software program (Horizon Image Viewer, version 1.5, OAISYS Medical Inc.) to locate bone landmarks on the femur and tibia from which standard measures of alignment (e.g. the Hip-Knee-Ankle (HKA) angle) and bone lengths could be computed. To assess the reliability of these alignment measurements, one hundred randomly assigned digital radiographs, representing two hundred limbs, were selected from the complete data set for a repeated analysis carried out two or more weeks after completion of the first measurements. Random effects two-way analysis of variance (ANOVA) models were applied to estimate the interclass and intraclass correlation coefficients (ICC), which correspondingly evaluated inter-reader and intra-reader reliability for each of the angles and bone lengths. High reliability measures were obtained for the HKA angle (inter-reader reliability: ICC=0.995 (95% CI, 0.994–1); intra-reader reliability: ICC= 0.998 (95% CI, 0.998–1)). Reliability for additional angles between the femur and tibia ranged from 0.839 to 0.993 (inter-reader reliability) and 0.908 to 0.998 (intra-reader reliability). High reliability measures were also obtained for bone lengths (inter-reader reliability: ICC from 0.993 to 0.995; intra-reader reliability: ICC from 0.994 to 0.995). Each of the lower limb alignment and bone length measurements were highly reliable. The outcome supports the use of computer software programs and software tools for analysis of lower limb frontal plane alignment.