Acute mastitis is an endemic disease in suckler ewes in the UK, with major implications for both farm sustainability and sheep health and welfare. Chronic mastitis, characterised by the presence of an intramammary mass (IMM), is highly correlated with occurrence of acute mastitis. Intramammary masses are believed to be polymicrobial, with a maturation and rupture cycle, resulting in an under-detection of their incidence within a flock. The aim of this study is to determine the role of intramammary masses in the persistence of bacterial strains in milk, and how the prevalence of these strains changes over time. During a two-year longitudinal study of six suckler sheep flocks, milk was collected from 89 ewes on four occasions. Ewes were defined as having an IMM, acute mastitis or clinically healthy. Milk samples were cultured aerobically on sheep blood agar (SBA) and each morphologically different isolate was selected for bio-typing. Matrix-assisted laser desorption/ionization time-of flight (MALDI-ToF) mass spectrometry was carried out on each isolate, to obtain a distinct mass spectrum and where possible a species and strain identification. Mass spectra were used to cluster isolates on similarity. Biological and technical replicates of known isolates were used to determine a threshold Euclidean distance; clusters below this value were considered to be the same bacterial strain. The clusters will be used to investigate persistence of bacterial strains within ewes and transmission between ewes and to link persistence and transmission events to the disease state of the ewe.