The quality of tree nuts can be influenced by their harvest time, cultivar and seasonal growing conditions. Some macadamia cultivars retain mature nuts in the tree for several months before fruit abscission and this can result in variations in harvest time. It is not known how the variations in harvest time affect the quality and shelf life of macadamia cultivars. We investigated how harvest time and cultivar influence the quality and shelf life of macadamia kernels. We tested the shelf life of kernels using two techniques: (1) a long-term shelf-life method; and (2) a short-term accelerated-aging method that assessed oxidative stability based on hexanal concentrations. Cultivars HAES 344 (344) and Hidden Valley A16 (A16) were harvested in May and late-abscising nuts of cultivar A16 were also harvested in September. Peroxide values, free fatty acid levels and fatty acid composition of kernels in the long-term experiment were measured after 0, 6, 12, 15 and 18 months of storage at 25 °C. Kernels in the short-term accelerated aging experiment were incubated at 45 °C, and hexanal concentrations were measured before incubation and after 6, 12 and 18 days. Kernels from late-abscising nuts (September) had much higher free fatty acid levels after 18 months of storage than kernels from early-harvested nuts (May). Cultivar A16 (September) kernels had significantly higher hexanal concentrations than cultivar 344 (May) and A16 (September) kernels at 12 and 18 days of incubation. The accelerated aging technique could detect differences in oxidative stability between different batches. Nuts harvested in September had shorter shelf life by about 3–6 months than those harvested in May, based on both predicted shelf life and actual time taken to reach acceptable-quality limits. Therefore, we recommend harvesting macadamia nuts as soon as possible after maturity to minimise quality losses.