The objective of this study was to evaluate the suitability of 6 handheld meters for measuring glucose in dairy cows [Contour Next (CT), FreeStyle Precision Neo (FS) Nova Max Plus (NM), Aga Matrix (AM), Precision Xtra (PX), and Accu-Chek Aviva Plus (AC)]. Whole blood samples from 97 Jersey cows (71 for CT) were measured in triplicate immediately after collection using the 6 handheld glucose meters. Statistical analysis was performed using Medcalc v.18. Plasma glucose concentration by hexokinase enzymatic methodology was considered the reference methodology. Based on the intra-assay coefficient of variation (CV), precision varied across handheld glucose meters: AC (2.9%), CT (6.1%), PX (6.2%), FS (7.3%), AM (8.3) and NM (8.3%). Pearson correlation coefficients between the reference methodology and handheld glucose meters were 0.53 for AM, 0.67 for NM, 0.79 for FS, 0.82 for CT, 0.83 for PX and 0.85 for AC (P < 0.0001). Bland-Altman plots between each meter and the reference methodology resulted in a negative bias (FS = −4.2 mg/dL; CT = −10.7 mg/dL) or a positive bias (PX = 5.9 mg/dL; AM = 7.0 mg/dL; NM = 10.4 mg/dL; AC = 11.8 mg/dL). All handheld glucose meters fell within acceptable limits of agreement (1.96SD) at least 95% of the time (PX = 96%; AC = 96%; CT = 95%; AM = 95%; NM = 95%), except by FS (94%). Optimized thresholds were calculated by receiver operating characteristics (ROC) analysis using ≥60 mg/dL as the thresholds for hyperglycemia. Adjusted thresholds were 47.0 mg/dL for CT (Sensibility [Se] = 92.1%, Specificity [Sp] = 72.7%), 51.3 for FS mg/dL (Se = 90.4%, Sp = 57.8%), 64.5 mg/dL for PX (Se = 76.9%, Sp = 73.3%), 69.7 mg/dL for AM (Se = 50.0%, Sp = 75.6%), 68.3 mg/dL for NM (Se = 73.1%, Sp = 60.0%), and 71.3 mg/dL for AC (Se = 75.0%, Sp = 75.6%). According to area under the ROC curve (AUC), CT (0.835) had the highest accuracy, followed by AC (0.799), PX (0.790), FS (0.751), NM (0.690), and AM (0.616). CT and AC had a discriminatory power comparable to a previously validated handheld meter (PX), but precision was better for AC. For dairy cows, most meters will need be used with optimized threshold or be calibrated to improve accuracy.