Different concentrations of nutrients and organic matter in water were assumed to be reflected in differences in root length of duckweeds. This parameter might thus be used for biological indication of water trophic status in 12 small village ponds in South Bohemia (The Czech Republic) during the development of duckweed (Lemnaceae) communities. We assessed the concentrations of total N, NO3-N, NO2-N, NH4-N, total P, PO4-P, K+, Ca2+, Mg2+, and Na+ in water and of N, P, K, Ca, Mg, and Na in the duckweed dry matter in order to see at what concentrations nutrients affect root length in Lemna minor and Spirodela polyrrhiza. The root length of Spirodela polyrrhiza and Lemna minor in pond biotopes was found to be controlled by concentration of phosphate in water and by BOD5, i.e., by oxygen availability to roots. These facts were confirmed by the negative correlation between root length and phosphorus concentration in plant dry matter: r = -0.75 and -0.92 for Lemna minor, and -0.73 and -0.65 for Spirodela polyrrhiza on two sampling dates. In addition, cultivation experiments were carried out with duckweeds originating from the ponds. These experiments have confirmed that root length of Lemna minor and Spirodela polyrrhiza is controlled by the concentrations of phosphorus and oxygen in water.