With increasing world interest in honey characterization various studies have been carried out relating physicochemical parameters to botanical and geographical origins. Ash content is one of these parameters that has been associated with floral sources of honey samples. However, little attention has been given to the determination of how much of the variability in ash contents is caused by contributions of different floral sources or honey samplings compared to the variations caused by analytical errors. So, in this work, a two stage hierarchical statistical design is employed to quantify the sizes of these different variational sources. An extremely significant effect on ash content owing to floral origin of Eucalyptus and Citrus honey samples was found. A smaller but significant variation was also observed for different samples from the same floral source. This sampling variance can be associated with different environmental and geographical conditions, different technological aspects of production and processing of honeys since the honey samples were obtained from various beekeepers and collected in diverse regions of São Paulo State, Brazil. Considering that the variation owing to floral source is much larger than the one observed for sampling and analysis errors, ash content could be used to characterize these two types of monofloral honeys.