Honey is a popular sweetener that makes significant contributions to human nutrition. The aim of this study was to analyze and comparespecific properties relating to honey quality and biological value such as total phenolic content, color, color intensity, proline and otherquality parameters like moisture content, electrical conductivity, 5-hydroxymethylfurfural, diastase activity in various honeys directly frombeekeepers and from markets. In total fiftysix honey samples directly from Czech and Slovak beekeepers (n=25) and from markets inthe Czech Republic (n=31) were collected during 2018. The determined parameters varied depending on the botanical and geographicalorigin of honeys. The identified most significant differences (p < 0.01) between the analyzed beekeepers and market honeys in color,color intensity, total phenolic content, moisture content and 5-hydroxymethylfurfural point to the different properties and impact of honeyprocessing on honey composition. In several parameters, like color, color intensity, total phenolic content, proline content and electricalconductivity a very good correlations with botanical origin of honey were observed and beside the melissopalynological analysis theseparameters participated on characterization and authentication of unifloral honeys. Normally, there is a positive correlation between colorand electrical conductivity, however we have also confirmed that the color of honey also depends on the proline content. The results ofthis study confirmed that the origin (beekeeper/market) and botanical source of honeys (unifloral/nectar/honeydew) had effect on theirquality and biological value.