Background Stair ascent mechanics change with age, but little is known about the differing functional demands of transitioning and continuous ascent. Work investigating the risky transition from gait to ascent is sparse, and the strategies that older adults adopt to achieve these demanding tasks have not been investigated. Methods This study compared the biomechanics of a 2-step transitional (floor-to-step2) and continuous ascent cycle (step1-to-step3) and investigated the role of limb preference in relation to dynamometer-derived knee strength during this transition. A biomechanical analysis of 36 women (60–83 years) ascending a 3-step staircase was conducted. Findings The 2-step transitioning cycle was completed quicker, with a larger range of motion, increased forces, larger knee flexor and dorsiflexor moments and ankle powers (P ≤ 0.05), but reduced hip and knee flexion, smaller hip extensor moments and hip and knee powers compared to continuous ascent. During the transition, 44% of the participants demonstrated a consistent limb preference. In these cases large between-limb extensor strength differences existed (13.8%) and 71% of these participants utilised the stronger limb to execute the 2-step transitional cycle. Interpretation The preferential stronger-limb 2-step transitioning strategy conflicts with previous recommendations of a stronger lead limb for frail/asymmetric populations. Our findings suggest that most healthy older women with large between-limb differences utilise the stronger limb to achieve the considerable propulsion required to redirect momentum during the 2-step transition. The biomechanical demands of ascent, relative to limb strength, can inform exercise programmes by targeting specific muscle groups to help older adults maintain/improve general functioning.