Introduction: Diabetes mellitus (DM) is one of the most prevalent diseases worldwide. The objective of this study was to find out under what preanalytical conditions routine and diagnostic glucose tests are performed across Spanish laboratories; and also what criteria are used for DM diagnosis. Materials and methods: An online survey was performed by the Commission on Quality Assurance in the Extra-Analytical Phase of the Spanish Society of Laboratory Medicine (SEQC-ML). Access to the questionnaire was available on the home page of the SEQC-ML website during the period April-July 2018. Data analysis was conducted with the IBM SPSS© Statistics (version 20.0) program. Results: A total of 96 valid surveys were obtained. Most laboratories were in public ownership, serving hospital and primary care patients, with high and medium workloads, and a predominance of mixed routine-urgent glucose testing. Serum tubes were the most used for routine glucose analysis (92%) and DM diagnosis (54%); followed by lithium-heparin plasma tubes (62%), intended primarily for urgent glucose testing; point-of care testing devices were used by 37%; and plasma tubes with a glycolysis inhibitor, mainly sodium fluoride, by 19%. Laboratories used the cut-off values and criteria recognized worldwide for DM diagnosis in adults and glucose-impaired tolerance, but diverged in terms of fasting plasma glucose and gestational DM criteria. Conclusion: Preanalytical processing of routine and DM diagnostic glucose testing in Spain does not allow a significant, non-quantified influence of glycolysis on the results to be ruled out. Possible adverse consequences include a delay in diagnosis and possible under-treatment.