Stingless bee (Hymenoptera: Apidae: Meliponinae) honey is a dark black coloured nutritious and medicinally valued substance, produced by stingless bee species by using the nectar and pollen of blossoms of various plant species. Stingless bee honey exhibits variation in its physical properties, chemical composition and nutritional quality. Therefore, present investigation was conducted to assess the physical parameters and chemical constituents in the stingless bee honey collected from Mysore, Chamarajanagar and Kodagu districts of southern Karnataka. Results revealed significant differences with respect to the physical parameters and chemical constituents between the stingless bee honey samples. The pH ranged from 3.42 to 3.55, electrical conductivity ranged from 0.668 to 1.412 mS/cm, specific gravity was in between 1.35 and 1.38 g/cm2, absorbance at 660 nm was 0.39 to 1.696. The turbidity ranked between 0.09 to 0.38 NTU and the moisture with the honey was in between 30.85 to 40.8 g/100 g. Moreover, the chemical parameters such as glucose content in the honey was ranged between 11.0 to 17.0 g/100 g, fructose was 30.0 to 40.0 g/100 g with protein 237.14 to 595.48 mg/100 g. However, the total dissolved solids were ranged in between 285.2 and 613.6 in the honey samples. Further, minerals like, calcium, magnesium, sodium, potassium, iron, copper, chromium, zinc, manganese and phosphorous content in terms of parts per million did vary significantly between the honey samples collected from Mysore, Chamarajanagar and Kodagu districts of southern Karnataka. Furthermore, vitamins such ‘C’, ‘B6’, thiamine, riboflavin, niacin, biotin and lipoic acid were also indicated significant variations in the honey samples. Thus, physical parameters and chemical constituents are unique to the honey sample, collected from various districts which likes at different agro-climatic regions and demonstrated the influence of locally available flora and climate of that geographical region. During the present investigation, region specific uniqueness of stingless bee honey of southern Karnataka was recorded and fulfilled the requirement of honey analysis for quality and human consumption as like Apis species honey.