The aim of this study was to test young-of-the-year (YOY) assemblage applicability for the ecological quality assessment on the basis of samples from 55 sites scattered across the entire Czech Republic. European Fish Index (EFI) values estimated with YOY and adults were positively correlated, indicating that YOY and adult sampling is comparable. YOY bias the EFI toward significantly lower values, however, adults also lead to underestimated EFI values on some occasions. Relative differences between EFI YOY and EFI adults show almost normal distributions and indicate that there is no detectable systematic bias caused by environmental conditions or intensity of sampling. As shown in the Elbe River example, the reason for lower EFI YOY values could be found in a longer period of detection of evident changes in adult fish assemblages, reflecting longevity of adult fishes or regular stocking. YOY sampling was suggested as a useful method for assessing river ecological quality, with the ability to provide a sensitive response to the habitat structure regardless of the effect of stocking or river size.