Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination, with poly-ubiquitination (Ub) of MHC II directing it away from the plasma membrane. The MHC II poly-Ub chain that dictates the fate and function of ubiquitinated MHC II has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary dendritic cells (DCs) and B cells. Analysis was conducted for endogenous H2-A and H2-E molecules immunoprecipitated from primary cells. We show that the ubiquitination of both I-A and I-E expressed by DCs and B cells is dependent on the E3 Ub ligase MARCH1. Comprehensive Ub chain analysis reveals MARCH1-dependent poly-Ub associated with MHC II is composed of Ub chains with branched K11/K63 linkages.