OBJECTIVE: Flexural strength values of multilayer zirconia may vary depending upon the specimen dimension, layers distribution, especially the layer in maximum tension side, i.e., loading configuration. Although the previous studies have examined flexural strength of the separate layers in one zirconia disc, capturing flexural strength across all layers of multilayer zirconia is still challenging yet important for their clinical indications. The objective of this study aimed to evaluate the flexural strength of multilayer graded translucent zirconia by three loading configurations with the full thickness of layers. MATERIALS AND METHODS: Four types of zirconia materials were selected namely KATANA Yttria Multi Layered (KATANA YML, Kuraray Noritake), IPS e.max ZirCAD Prime (ZirCAD Prime, Ivoclar Vivadent), and Origin Beyond + Hybrid (Origin Beyond, B&D Dental Technologies) multilayer zirconia, as well as monolithic InCoris translucent zirconia (inCoris TZI, Dentsply Sirona). The bar specimens were prepared by sectioning the zirconia discs with diamond blade to get the dimensions of 31.0 mm x 4.6 mm x 11.0 mm for KATANA YML and Origin Beyond, 42.0 mm x 6.4 mm x 13.1 mm for ZirCAD Prime, and 31.0 mm x 4.2 mm x 10.0 mm for inCoris TZI. The specimens were sintered with a Zircar high temperature furnace by following the protocols of manufacturers accordingly. The four longitudinal surfaces of all bar specimens for all materials were polished with diamond discs in a sequence of 125, 70, 45, 15, and 6 mm, and down to 0.5 mm finish. All polished specimens were subjected to the annealing treatment at 1000 oC for 15 min before mechanical testing. The three-point bend testing with three loading configuration was performed for flexural strength on the Universal Testing Machine (Instron 5566A) with a loading rate of 0.5 mm/min. The fracture surfaces of these bar specimens were examined by a field emission scanning electron microscope. The yttria (Y2O3) and hafnia (HfO2) concentrations were analyzed by energy disperse spectroscopy on polished surfaces. RESULTS: The highest mean 3 point bending flexural strength for all layers direction was for inCoris TZI with 786.55 MPa, whereas the lowest for Origin Beyond with 505.36 MPa. The inCoris TZI had overall highest flexural strength with vertical (enamel or dentin layer in tension) of 845.18 MPa. ZirCAD Prime had overall highest flexural strength with the loading configuration of dentin layer in tension of 906.22 MPa while Origin Beyond had the lowest flexural strength of 686.79 MPa. Under the loading configuration of enamel layer in tension, the flexural strength of all three multilayer materials ZirCAD Prime, Origin Beyond and KATANA YML had no significant difference, having a strength value range of 498-504 MPa. The dentin layer had the lowest yttria concentration while the enamel had the largest one, and the transition layer was between them. The distribution of hafnia concentration of different layers for all multilayer materials was consistent. CONCLUSION: Within the limitations of this in vitro study, we can conclude that material, and loading configurations as well as the interaction between material and loading configurations had a significant effect on flexural strength of multilayer zirconia. ZirCAD Prime showed a significantly higher flexural strength than KATANA YML whereas Origin Beyond had the lowest flexural strength. The flexural strength of dentin layer in tension was significantly higher than all layers in tension, while the flexural strength of all layers was significantly higher than enamel layer in tension. The yttria concentration of multilayer zirconia gradually increased from dentin layer to enamel layer whereas the hafnia concentration was consistent across different layers. These findings in this study could enrich the knowledge of clinicians when selecting the multilayer zirconia materials in the clinical settings.