Yersinia enterocolitica is a zoonotic enteropathogenic bacterium that can cause acute gastroenteritis and mesenteric lymphadenitis. Although Y. enterocolitica is common in animals, food, and the environment, the reservoirs and transmission routes of this pathogen are still not fully understood. The aim of our study was to determine the prevalence of Y. enterocolitica in seafood in Germany, because only limited data are available on that topic. Seafood samples were purchased from retail shops in Berlin, Germany and examined for the presence of Y. enterocolitica by cold enrichment followed by cultivation on selective agar. Presumptive Y. enterocolitica isolates were analyzed by biotyping, serotyping, and antimicrobial susceptibility testing. The total prevalence of Y. enterocolitica in seafood samples was 2.7% (6 of 220 samples). Mussel (2 of 90), shrimp (1 of 89), and cephalopod (3 of 41) samples were positive for Y. enterocolitica. Three isolates were identified as serotype O:8, one was identified as serotype O:5,27, and two samples did not belong to any investigated serotypes. The presence of the virulence-associated genes ail, inv, and ystB was studied by multiplex PCR. Four of the six isolates contained inv and ystB, one produced no positive results for the analyzed genes, and one contained only ystB. All Y. enterocolitica isolates were susceptible to cefotaxime, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, tetracycline, and trimethoprim. Resistance was observed to cephalothin (83.3% of isolates), amoxicillin (83.3%), and ampicillin (50.0%). This study provides the first comprehensive analysis of Y. enterocolitica in retail seafood in Germany. The prevalence found in these seafood samples was comparatively low, and all isolates belonged to biotype 1A. However, seafood contaminated with Y. enterocolitica may pose a risk to consumer health because the pathogenic potential of biotype 1A strains is still being debated.