Left atrial strain (LAS) is the most promising technique for assessment of diastolic dysfunction but few data are available in neonates. Our aim was to assess feasibility and reproducibility, and to provide reference ranges of LAS in healthy neonates in the first 48 h of life. We performed one echocardiography in 30 neonates to assess feasibility and develop a standard protocol for image acquisition and analysis. LAS reservoir (LASr), conduit (LAScd) and contraction (LASct) were measured. We performed echocardiography at 24 and 48 h of life in an unrelated cohort of 90 neonates. Median (range) gestational age and weight of the first cohort were 34.4 (26.4–40.2) weeks and 2075 (660–3680) g. LAS feasibility was 96.7%. Mean (SD) gestational age and weight of the second cohort were 34.2 (3.8) weeks and 2162 (833) g. Mean (SD) LASr significantly increased from 24 to 48 h: 32.9 (3.2) to 36.8 (4.6). Mean (SD) LAScd and LASct were stable: −20.6 (8.0) and −20.8 (9.9), −11.6 (4.9) and −13.5 (6.4). Intra and interobserver intraclass correlation coefficient for LASr, LAScd and LASct were 0.992, 0.993, 0.986 and 0.936, 0.938 and 0.871, respectively. We showed high feasibility and reproducibility of LAS in neonates and provided reference ranges.