Due to potential lethality of healthcare-associated sepsis (HAS), a low threshold for blood culturing and antimicrobial therapy (ABT) initiation is accepted. We assessed variability in the trigger for blood culturing between three neonatal intensive care units. A multicenter prospective cohort study was conducted. In newborns with suspicion of HAS, 10 predefined clinical signs, nosocomial sepsis (NOSEP) score, C-reactive protein, ABT initiation, and risk factors were registered at time of culturing. Outcome was lab-confirmed HAS, defined according to the NeoKISS-criteria. Two hundred ninety-nine suspected HAS episodes were considered in 212 infants, of which 118 had birth-weight ae<currency> 1500 g; proportion of lab-confirmed HAS per suspected episode was 30/192 (center 1), 28/60 (center 2), and 8/47 (center 3) (p < 0.001). Median C-reactive protein and number of clinical signs at time of culturing differed between centers 1, 2, and 3 (respectively 11 vs. 5 vs. 3 mg/L, p = 0.001; 1 sign [IQR 0-2, center 1] vs. 3 signs [IQR 2-4, centers 2 and 3], p < 0.001). Median NOSEP score at time of culturing was 5 (IQR 3-8, center 1), 5 (IQR 3-9, center 2), and 8 (IQR 5-11, center 3) (p = 0.016). Difference in ABT initiation was noticed (82 vs. 93 vs. 74%, p = 0.05). Conclusion: Center heterogeneity in sampling practice is substantial. Optimizing sampling practice can be recommended.