Low-abundance milk proteins are of interest because of their diverse bioactivity. The objective of this study was to characterize the low-abundance protein profile within the whey fraction of milk produced by 2 dairy cattle breeds. A 7-d trial was conducted with 6 Jersey (80 ± 49 DIM) and 6 Holstein (75 ± 21 DIM) cows paired by DIM and parity, housed in the same tie-stall barn. Cows were all maintained on the same TMR diet and were fed individually to determine daily DMI. Milk samples were collected at a.m. and p.m. milking during the experiment. Milk composition (protein, fat, milk urea nitrogen (MUN), SCC) was determined in all samples collected and subsamples were collected and stored at −80C for low-abundance protein analysis. Milk samples for low-abundance protein analysis were thawed, pooled within animal and a mammalian protease inhibitor was added to each sample before centrifugation to isolate the whey fraction. Samples were depleted of casein and separated by SDS-PAGE. Samples were excised and subjected to a tryptic digest followed by LC-MS/MS analysis on a linear ion trap (LTQ)-Orbitrap Mass Spectrometer (MS). Product ion spectra were searched using SEQUEST on Proteome Discoverer 1.4 against a curated Bovine database. A linear mixed model was used to perform a repeated measures analysis on milk parameters and DMI in SAS (9.4). DMI (P = 0.0108), milk protein % (P = 0.0021), protein yield (kg/d; P = 0.0068) and fat % (P = < 0.001) were different between breeds whereas MUN (P = 0.86) and SCC (P = 0.59) were not different between breeds. MS analysis identified 947 proteins including over 45 proteins present at significantly different peptide counts between breeds (P < 0.05) with fold differences in peptide counts ranging from 0.2 to 6.5. Some known bioactive proteins were present at significantly different levels, including lactotransferrin (P = 0.0026) and complement C2 (P = 0.0001), whereas other known bioactive proteins including osteopontin (P = 0.17) and lactoperoxidase (P = 0.29) were present at similar levels in both breeds. This work provides insight into the low-abundance protein composition of milk produced by 2 dairy breeds.