Rapid and reliable determination of blood glucose concentration is essential during the neonatal period to prevent adverse neurodevelopmental outcome from hypoglycaemia. Despite their unreliability, reagent strip methods continue to be used extensively in neonatal nurseries due to their rapidity and convenience. Recently, a new portable laboratory standard technique has been introduced (HemoCue B-Glucose system) for whole blood glucose determination. It is particularly suitable for near-patient testing in neonatal units. This new method, as well as other established methods of whole blood (Yellow Springs Instrument (YSI) and a hexokinase method on Cobas Bio), and plasma (Kodak Ektachem) glucose measurement, were therefore evaluated for their accuracy and concordance of measurements taken in the neonatal period. There were substantial discrepancies among the four methods of glucose measurement with wide limits of agreement between these methods. The glucose concentrations measured by HemoCue and YSI (n = 206), HemoCue and hexokinase (n = 113), HemoCue and plasma glucose on Ektachem (n = 69) and hexokinase and Ektachem (n = 66) were likely to differ by -29 to +61%, -23 to +56%, -36 to +65%, and -19 to +30%, respectively. Even the laboratory methods of blood glucose determination, therefore, can not be used interchangeably. Using a model based approach, the probabilities of “discordant” classification as hypo- or normo-glycaemia were estimated to be 6.8%, 6.5%, and 7.1% between HemoCue and YSI, HemoCue and hexokinase on Cobas Bio, and HemoCue and Ektachem analysers, respectively. In view of these low probabilities of discordant classification with other glucose analysers, the HemoCue system may offer a reasonable compromise between bedside and laboratory blood glucose estimations in neonates.