Mānuka (Leptospermum scoparium) is valued for its unique bioactive honey, but relatively little is known about mānuka nectar and the factors modulating its production. In this study the compositional variability of nectar collected throughout mānuka floral development was measured in glasshouse-cultivated plants. The total yield of nectar was estimated by rinsing flowers with water and measuring total sugars (°Brix). The concentration of total sugars and of dihydroxyacetone (DHA), the precursor to methylglyoxal and the key mānuka honey bioactive, increased through floral development and maximised just prior to initiation of flower degradation. Similarly, the patterns of several mānuka honey authenticity markers including lepteridine, leptosperin and 3-phenyllactic acid also followed a similar trend through flower development, suggesting that their biosynthesis was associated with nectar production. Leaf exudates produced as a result of scale insect infestation of some mānuka plants were deficient in the key markers DHA, leptosperin and lepteridine, but did contain 4-hydroxyphenyllactic acid. This study suggests that the predominant key markers of genuine mānuka are biosynthesised in the floral tissues of the plant rather than elsewhere. For robust analysis and comparison of mānuka nectar yield and composition, sampling needs to take into account the floral developmental stage of the flowers sampled.