Locomotion imposes some of the highest loads upon the skeleton, and diverse bone designs have evolved to withstand these demands. Excessive loads can fatally injure organisms; however, bones have a margin of extra protection, called a ‘safety factor’ (SF), to accommodate loads that are higher than normal. The extent to which SFs might vary amongst an animal's limb bones is unclear. If the limbs are likened to a chain composed of bones as ‘links’, then similar SFs might be expected for all limb bones because failure of the system would be determined by the weakest link, and extra protection in other links could waste energetic resources. However, Alexander proposed that a ‘mixed-chain’ of SFs might be found amongst bones if: 1) their energetic costs differ, 2) some elements face variable demands, or 3) SFs are generally high. To test if such conditions contribute to diversity in limb bone SFs, we compared the biomechanical properties and locomotor loading of the humerus and femur in the tiger salamander (Ambystoma tigrinum). Despite high SFs in salamanders and similar sizes of the humerus and femur that would suggest similar energetic costs, the humerus had lower yield stresses, higher mechanical hardness, and larger SFs. SFs were greatest in the anatomical regions where yield stresses were highest in the humerus and lowest in the femur. Such intraspecific variation between and within bones may relate to their different biomechanical functions, providing insight into the emergence of novel locomotor capabilities during the invasion of land by tetrapods.