This paper had as objective the isolation of different bacteria, which cause mastitis and, simultaneously, make emphasis on the differentiation of Mycoplasma species, taken from stored milk samples and individual cows. The animals and the milk were provided by farms from the Boyaca ́s high altitude plateau. The process was carried out through a convenience probabilistic sampling and also a descriptive, longitudinal and retrospective research, during 2010 and 2012. The researches took into account 30 technically managed milking farms, each with approximately 50 milking cows. Streptococcus agalactiae was isolated from 9.1%, being the most detected pathogen. The mean Staphylococcus aureusisolates was of 6.6%. Mycoplasma bovis was not found in any sample during the three year period of the research, while M californicum was recovered from 14 samples (1.5%) during this time lapse and M. alcalescens was detected in 31 samples (3%). Acholeplasma spp. was isolated from 19 samples (1.4%). This research corresponds to first report of Mycoplasma spp. Isolation from milk samples at the Altiplano Boyacense Colombia.