Background: Hyperbilirubinemia (HBN) can cause myocardial injury in neonates. Advancement in myocardial deformation imaging allows the detection of subclinical changes in myocardial contractility.The present study aimed to evaluate the changes in left ventricular contractility in newborns with hyperbilirubinemia by 2D speckle tracking imaging (STI).Methods: A group of 134 neonates who reached the diagnostic level of HBN as the HBN group was selected. The control group included 56 healthy newborns. The interventricular septum, anterior partition, anterior wall, sidewall, posterior wall, and inferior wall were separated into the basal, middle, and apical segments. In each segment, speckle tracking analysis was performed in the subintimal, middle, and subadventitial myocardium. The overall longitudinal strain of the myocardium in different ventricular walls and segments and global longitudinal strain (GLS) were computed. At the same time, the laboratory results of blood gas analysis, blood routine tests, liver function, and myocardial enzyme spectrum in HBN neonates were collected and correlated with the left ventricular stratified strain parameters.Results: The gradient of the left ventricular GLS had the same characteristics in both groups of newborns.There was a decreasing trend of longitudinal strain (LS) from the intima to the adventitia (i.e., GLSendo > GLSmid > GLSepi). This gradient was also present in stratified LS in each myocardial segment (P<0.001).The LS showed an increasing trend from the basal to the apical segment (P<0.001). The LS of the ventricular septum, anterior wall (or anterior septum), inferior wall, lateral wall, and posterior wall showed a decreasing trend (P<0.001). Stratified strain parameters of the ventricular wall (i.e., the 3-layer myocardium: LSendo-SEPT, LSmid-SEPT, and LSepi-SEPT) were all significantly lower in the HBN group than in the control group (P=0.