© 2019, The Author(s). Eukaryotes are important components of ecosystems in wastewater treatment processes. However, little is known about eukaryotic community in anaerobic wastewater treatment systems. In this study, eukaryotic communities in an up flow anaerobic sludge blanket (UASB) reactor treating domestic sewage during two years of operation were investigated using V4 and V9 regions of 18S rRNA gene for amplicon sequencing. In addition, activated sludge and influent sewage samples were also analyzed and used as the references for aerobic eukaryotic community to characterize anaerobic eukaryotes. The amplicon sequence V4 and V9 libraries detected different taxonomic groups, especially from the UASB samples, suggesting that commonly used V4 and V9 primer pairs could produce a bias for eukaryotic communities analysis. Eukaryotic community structures in the UASB reactor were influenced by the immigration of eukaryotes via influent sewage but were clearly different from the influent sewage and activated sludge. Multivariate statistics indicated that protist genera Cyclidium, Platyophrya and Subulatomonas correlated with chemical oxygen demand and suspended solid concentration, and could be used as bioindicators of treatment performance. Uncultured eukaryotes groups were dominant in the UASB reactor, and their physiological roles need to be examined to understand their contributions to anaerobic processes in future studies.