Casein phosphopeptides are multiphosphorylated milk peptides, which can have anticariogenic activity and improve mineral absorption by binding bivalent metal ions. The present study investigated phosphopeptides in kefir because fermentation may lead to their enhanced release from milk proteins. After selective enrichment by hydroxyapatite extraction, phosphopeptides and their phosphorylation degree were identified by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) before and after enzymatic dephosphorylation. Peptide structures were determined by ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) revealing 27 phosphopeptides in kefir, including nine peptides containing the motif pSpSpSEE, which binds minerals most efficiently. The majority (18) of phosphopeptides were derived from β-casein, but only three were derived from the most abundant milk protein αs1-casein. After simulated gastrointestinal digestion, MALDI-TOF-MS analysis detected eight putative phosphopeptides in kefir, four of which were assigned by UHPLC-ESI-MS/MS to αs2-casein124-133, αs2-casein137-146, β-casein30-40, and κ-casein147-161. These results indicate that kefir is a good dietary source of multiphosphorylated peptides.