A significant portion of equine lameness is localized to the stifle joint. Effective cartilage repair strategies are largely lacking, however, recent advances in surgical techniques, biomaterials, and cellular therapeutics have broadened the clinical strategies of cartilage repair. To date, no studies have been performed directly comparing neonatal and adult articular cartilage from the stifle across multiple sites. An understanding of the differences in properties between the therapeutic target cartilage (i.e., adult cartilage) as well as potential donor cartilage (i.e., neonatal cartilage) could aid in selection of optimal harvest sites within a donor joint as well as evaluation of the success of the grafted cells or tissues within the host. Given the dearth of characterization studies of the equine stifle joint, and in particular neonatal stifle cartilage, the goal of this study was to measure properties of both potential source tissue and host tissue. Articular cartilage of the distal femur and patella (P) was assessed in regards to two specific factors, age of the animal and specific site within the joint. Two age groups were considered: neonatal (<1 week) and adult (4-14 years). Cartilage samples were harvested from 17 sites across the distal femur and patella. It was hypothesized that properties would vary significantly between neonatal and adult horses as well as within age groups on a site-by-site basis. Adult thickness varied by site. With the exception of water content, there were no significant biochemical differences among sites within regions of the distal femur (condyles and trochlea) and the patella in either the adult or neonate. Neonatal cartilage had a significantly higher water content than adult. Surprisingly, biochemical measurements of cellularity did not differ significantly between neonatal and adult, however, adult cartilage had greater variance in cellularity than neonatal. Overall, there were no significant differences between neonatal and adult glycosaminoglycan content. Collagen per wet weight was found to be significantly higher in adult cartilage than neonatal when averaged across all levels. In terms of biomechanical properties, aggregate modulus varied significantly across the condyles of adult cartilage but not the neonate. Neonatal cartilage was significantly less permeable, and the Young's modulus of neonatal cartilage was significantly higher than the adult. The tensile strength did not vary in a statistically significant manner between age groups. An understanding of morphological, histological, biochemical, and biomechanical properties enhances the understanding of cartilage tissue physiology and structure-function relationships. This study revealed important differences in biomechanical and biochemical properties among the 17 sites and among the six joint regions, as well as age-related differences between neonatal and adult cartilage. These location and age-related variations are informative toward determining the donor tissue harvest site.