In this study, we estimated the applicability of length-weight relationship-based biomass calculations by comparison of body length of genus Polyarthra collected from different habitats. Through the comparison, we also tested availability of representative species-specific biomass value of Polyarthra which is often used without length measurement. Polyarthra samples were collected from rivers (Han River and Nakdong River) and reservoir (Paldang Reservoir), and the body length was measured for statistical comparison among habitats and biomass calculations using different equations suggested previously. According to the results, the body length of Polyarthra spp. was significantly different among sampling sites, and the necessity of body length measurement for rotifer species in each situation has been suggested rather than using the representative biomass values which is fixed without considering time and space. Comparison of suggested biomass calculations based on our measured Polyarthra body length, the equation suggested by McCauley showed more reasonable range of biomass values than that suggested by EPA. In addition, in order to calculate more accurate biomass, it is necessary to measure the body length of rotifers, at least more than 44 individuals to reduce error probability to less than 5% with 99% probability. However, since direct measurement of rotifers biomass is limited, it is considered that further analyses are required for more precise application of rotifer biomass of which has high variability due to complex morphologies and species-specific cyclomorphosis often induced by biotic and abiotic factors in the habitats.