A previous study from our laboratory, based on data collected during a large-scale field trial, found the osmotic resistance (ORT) of boar sperm to be positively correlated with in vivo fertility. The objectives of this study were to differentiate boars of diverse ORT and to assess the protein profile of their sperm and seminal plasma so as to identify potential biomarkers of fertility. Semen was collected at a commercial AI centre from 8 boars (6 collections; collection = replicate) and diluted to 30 × 10⁶ sperm/mL in Beltsville Thawing Solution. ORT was assessed by flow cytometry, using a viability assay (SYTO-16 and Propidium Iodide), post hypo-osmotic incubation for 20 min at 330, 165, 90 and 3 mOsm. Sperm protein profiles were investigated using ICM-MS (Intact Cell MALDI-TOF - Mass Spectrometry) on whole sperm cells while proteins from seminal plasma of the boars were assessed by MALDI-TOF. Protein Spectral Counting was also performed on sperm extracts and seminal plasma using geLC-MS/MS. ORT data was normalised against the 330 mOsm control and analysed using univariate procedures in SPSS (version 20). The effect of boar was employed in the final model, and deemed significant if P < 0.05. MALDI-TOF peaks were analysed using Progenesis MALDI software and deemed significant if P < 0.01 and had a fold change > 2. Boars were categorised into high, medium and low categories of ORT based on their ORT responsiveness at 165 mOsm. For sperm and seminal plasma, 17 and 20 proteins, respectively, ranging from 2 to 20 kDa differed between ORT categories (P < 0.01). The quantity of proteins such as fibronectin, thrombospondin, AQN-1, AQN-3, PSPI and PSPII in sperm were negatively associated with osmotic resistance, whereas, PRDX5 and alpha-mannosidase were positively correlated.