The objective of this study was to analyze factors affecting the prevalence of subclinical hypocalcemia on commercial dairies. Serum samples were collected from fresh dairy cows (n = 1,724) and analyzed for total Ca concentration (mg/dL). Time of sample collection and time of parturition were recorded. Other information collected included parity and postpartum Ca supplementation. A TMR sample was collected to determine prepartum diet mineral concentrations (including DCAD, regardless of source of supplemental anions). Data were analyzed using PROC Glimmix of SAS, using the binomial distribution option to generate LSMeans ± SE output describing percent subclinical hypocalcemia. Cows with serum Ca concentrations ≤8.5 mg/dL were considered subclinically hypocalcemic (SCH) and significance was determined at P ≤ 0.05. Fixed factors included parity, time of blood collection (0–6, 6–12, 12–36, 36–60, and >60 h post-calving), DCAD (<0, 0–11, 11+ mEq/100 g DM), dietary Ca (<0.75%, 0.75–1.50%, or >1.51% DM) and Ca supplementation (any form or method of Ca administered before blood sample collection). Parity, time of sample collection, DCAD and dietary Ca were analyzed for interactions with Ca supplementation. Parity influenced SCH (P < 0.0001), with increased prevalence of SCH occurring in 4+ parity cows. Time of sample collection affected SCH (P = 0.007). Cows fed negative DCAD diets prepartum had lower (13.84 percentage units) prevalence of SCH than those fed a prepartum diet with a DCAD greater than 11 mEq/100g DM (P = 0.008). Prepartum dietary Ca concentrations affected SCH (P = 0.04), with high Ca diets (>1.51%; 54.68 ± 4.41% SCH) tending to have lower prevalence of SCH than medium (0.75–1.5%; 60.37 ± 3.94% SCH) or low Ca (<0.75%; 65.08 ± 5.04% SCH) diets (P = 0.07 and P = 0.09 respectively). The interaction of prepartum dietary Ca and postpartum Ca supplementation significantly affected SCH (P = 0.03). These results indicate that numerous factors and their interactions affect serum Ca status in postpartum dairy cows and the prevalence of SCH.