The ideal number of blood samples to be obtained from peripheral veins (PVs) when differential time to positivity (DTTP) is being performed is an unresolved issue and most institutions obtain a single set. Our objective was to assess the number of proven central line-associated bloodstream infection (CLABSI) episodes that would have been recovered if blood had been cultured from one or two PVs. We performed a retrospective study in patients with proven CLABSI in which catheter lumens and two or more PV blood cultures were taken simultaneously. We calculated the number of episodes that would have been recovered if the culture of one or more PV blood cultures had been artificially eliminated. During a period of 4 years, we collected 60 episodes of proven CLABSI. Overall, if one PV culture had been eliminated in patients with two or three PV blood cultures, we would have documented 91.8% (p=0.362) and 96.9% (p>0.999) of episodes, respectively. If we had eliminated two PV blood cultures in patients with three PV blood cultures, 90.8% (p>0.999) of episodes would have been documented. When performing the DTTP technique to confirm CLABSI, a single paired PV blood culture was not associated with a significant number of missed CLABSI episodes.