Background Catheter-related bloodstream infection (CRBSI) is associated with increased morbidity, mortality, and cost of treatment in critically ill patients. A differential time to positivity (DTP) of 120 min or more between blood cultures obtained through the catheter vs. peripheral vein is an indicator of CRBSI with high sensitivity and specificity. However, it is no clear whether pooled sampling would be as efficient as individual sampling in order to reduce costs, contamination, or anemia. Methods This was a prospective diagnostic study conducted at the medical ICU and semi-ICU of Khon Kaen University’s Srinagarind Hospital in Thailand from May 2020 to November 2021. Fifty patients with triple-lumen central venous catheters (CVCs) who were clinically suspected of CRBSI were enrolled. 15 mL of blood was drawn through each catheter lumen, 10 mL of which was inoculated into three blood culture bottles, and the remaining 5 mL was pooled into a single bottle. Sensitivity, specificity, accuracy, and time to positivity of the pooled blood cultures were calculated using individual blood cultures as a reference. Results Of the 50 patients enrolled, 14 (28%) were diagnosed with CRBSI, 57.9% of whom were infected with gram-negative bacteria as the causative pathogen (57.9%). Extensively drug-resistant (XDR) Klebsiella pneumoniae was the most common organism. Sensitivity and specificity of the pooled blood sampling method were 69.23% (95% CI [0.44–0.94]) and 97.3% (95% CI [0.92–1.02]), respectively. The area under the ROC curve (AUC) was 0.83 (95% CI [0.68–0.99]). A paired T-Test to compare time to positivity of the pooled blood bottle and the first positive culture from the individual bottles indicated statistical significance (14.9 and 12.4 h, respectively). The mean difference was 2.5 [0.9–4.1] h, with a 95% CI and a p-value of 0.006. Conclusion Pooled blood sampling results in a lower sensitivity and longer time to positivity for CRBSI diagnosis in patients with triple-lumen CVCs than individual lumen sampling. Trial registration Retrospectively registered at Thai Clinical Trials Registry. The study was reviewed and approved on 08/03/2022. TCTR identification number is TCTR20220308002