Frequent outbreaks of infectious keratoconjunctivitis have been reported in wild Caprinae in Europe. While etiologic studies in the Alps indicate that the main etiologic agent is Mycoplasma conjunctivae, there are few reports from other mountain areas, such as the Pyrenees, where M. conjunctivae has never been reported. In 2006 and 2007, five adult Pyrenean chamois (Rupicapra pyrenaica; two males and three females) and one adult male European mouflon (Ovis orientalis musimon) were studied; they exhibited clinical symptoms of infectious keratoconjunctivitis such as blindness, corneal opacity, and ulceration. In three of the five chamois tested, and in the mouflon, Mycoplasma conjunctivae was identified from conjunctival swabs by means of a TaqMan(R) polymerase chain reaction based on the lipoprotein gene lppS. Cluster analysis indicated that the three southern chamois isolates form a cluster that is distinct from the mouflon isolate. This is the first report of M. conjunctivae in Pyrenean chamois, and it supports the hypothesis that M. conjunctivae also could be the main cause of infectious keratoconjunctivitis in areas other than the Alps, such as the Pyrenees.