Udder cleft dermatitis (UCD) or udder sores is a skin lesion, characteristically located around the anterior junction between the udder and abdomen of dairy cows. It is a worldwide problem in dairy herds with a large effect on animal welfare. The etiology and possible infectious origins of UCD are largely unknown; however, specific bacterial or parasitic causes are suggested in the literature. Therefore the aim of this study was to investigate the possible bacteriological, mycological, or parasitic involvement in clinically scored UCD lesions. Bacteriological culture was performed on subcutaneous tissue samples taken postmortem at a depth of 5 to 10 mm of 87 mild or severe UCD lesions or from the same place in healthy cows. Fungal culture was performed on a subset of 22 subcutaneous tissue samples of severe UCD postmortem. To investigate the superficial flora, swabs were taken from normal skin or skin lesions of 15 live animals equally divided over 3 groups: healthy skin or mild and severe UCD lesions. Histopathology, to describe and classify the lesions and to assess the presence of mites, fungi, or bacteria, was performed on 128 tissue samples, taken separately. In severe UCD lesions, Trueperella pyogenes and Bacteroides pyogenes were more frequently present in deep tissue layers and in superficial layers, compared with the same layers in mild UCD lesions or healthy skin. Culturing and histopathology indicated no sign of involvement of treponemes, Staphylococcus aureus, Escherichia coli, fungi, or mites in the UCD lesions. Histopathological examination showed that the majority of the lesions were characterized by chronic aspecific inflammation. Severe UCD lesions more frequently showed chronic active inflammation on histopathology, compared with mild UCD lesions. Due to the cross-sectional character of this study, it is difficult to differentiate cause and effect; however, future preventive and curative measures against UCD should take into account the chronic and anaerobic nature of this illness.