Blood gas and biochemistry reference intervals were established for 3 genetically distinct commercial varieties (CVs) of Hy-Line laying hens: 2 brown-egg layers (Hy-Line Brown, Hy-Line Silver Brown) and a tint-egg layer (Hy-Line Sonia) utilizing the i-STAT1 analyzer. Each respective variety of laying hen was sampled on a replicate cycle of 2 wk for a total of 6 replicates (35 to 46 wk of age). Blood samples were obtained in the production house from the brachial vein, and subsequently analyzed by the i-STAT1 portable device. i-STAT1 clinical analyzer reports blood gas and biochemistry values for the following parameters: pH, partial pressure of carbon dioxide (pvCO2, mm Hg), partial pressure of oxygen (pvO2, mm Hg), bicarbonate (HCO3, mmol/L), base excess (BE, mmol/L), saturation of oxygen on hemoglobin (sO2%), glucose (Glu, mg/dL), sodium (Na, mmol/L), potassium (K, mmol/L), total concentration of carbon dioxide (TCO2, mmol/L), ionized calcium (iCa, mmol/L), hematocrit (Hct % packed cell volume [PCV]), hemoglobin (Hb, g/dL). A total of 1,800 individual hen i-STAT1 records were utilized in the establishment of reference interval values for the 13 parameters between the 3 CVs. Statistical analysis via ANOVA and Tukey's test revealed significant line differences for all 13 blood gas and chemistry parameters measured, with particularly interesting results in iCa. The blood gas and chemistry parameters collected in this study will serve as reference intervals to set the framework for potential future correlations to genetic markers, physiological abnormalities, and production performance.