The objective of this longitudinal cohort study was to discover whether the milk microbiome of healthy bovine mammary glands differs from that of inflamed mammary glands during the first 150 d in milk (DIM). Aseptic milk samples were collected from each mammary quarter (n = 649) of cows in the UW-Madison dairy herd immediately before dryoff and twice within the first 14 DIM. Samples were cultured for bacterial growth and SCC was measured. Mammary quarters (n = 107) were enrolled to 4 cohorts based on microbiological status and somatic cell count (SCC) at dryoff and early lactation milk samples: LowNeg quarters (n = 81) had a SCC <100,000 cells/mL and no bacterial growth at all 3 samples; HighNegPre quarters (n = 17) had a SCC ≥150,000 cells/mL and no bacterial growth at the dryoff and first calving samples with a variable second calving sample; HighNegPost quarters (n = 6) had a SCC <100,000 cells/mL at the dryoff sample with a SCC ≥150,000 cells/mL at both calving samples and no bacterial growth at any of the 3 samples; HighPos quarters (n = 3) had bacterial growth and a SCC ≥150,000 cells/mL at all 3 samples. Milk samples were collected from all enrolled quarters weekly for SCC and an aseptic milk sample was collected every 28 d until 150 DIM for microbiological analysis. DNA was extracted from aseptic milk samples and PCR was performed with barcoded primers to the V4 region of the 16S rRNA gene; amplified PCR products were run on a gel, extracted, and sequenced on an Illumina MiSeq platform. Overall bacterial DNA load in our milk samples was low, as evidenced by only approximately 50% of milk samples that had visible DNA bands after PCR. Sequence data were analyzed in mothur. In samples that could be sequenced, Halomonas spp. were the most common operational taxonomic unit (OTU) at dryoff, calving, and through the first 150 DIM. Bacterial richness was similar among cohorts across time while Shannon diversity was greatest in LowNeg samples (mean = 2.93; P = 0.02). Differing microbial diversity in inflamed and uninflamed mammary quarters suggests that the milk microbiome may be associated with health status and change during lactation.