In this work, thermal cycling (T/C) reliability of anisotropic conductive film (ACF) flip chip assemblies having various chip and substrate thicknesses for thin chip-on-board (COB) packages were investigated. In order to analyze T/C reliability, shear strains of six flip chip assemblies were calculated using Suhir’s model. In addition, correlation of shear strain with die warpage was attempted.The thicknesses of the chips used were 180μm and 480μm. The thicknesses of the substrates were 120, 550, and 980μm. Thus, six combinations of flip chip assemblies were prepared for the T/C reliability test. During the T/C reliability test, the 180μm thick chip assemblies showed more stable contact resistance changes than the 480μm thick chip assemblies did for all three substrates. The 550μm thick substrate assemblies, which had the lowest CTE among three substrates, showed the best T/C reliability performance for a given chip thickness.In order to investigate what the T/C reliability performance results from, die warpages of six assemblies were measured using Twyman–Green interferometry. In addition, shear strains of the flip chip assemblies were calculated using measured material properties of ACF and substrates through Suhir’s 2-D model. T/C reliability of the flip chip assemblies was independent of die warpages; it was, however, in proportion to calculated shear strain. The result was closely related with material properties of the substrates. The T/C reliability of the ACF flip chip assemblies was concluded to be dominatingly dependent on the induced shear strains of ACF layers.