Biallelic MYO5B variants have been associated with familial intrahepatic cholestasis (FIC) with low serum gamma-glutamyltransferase (GGT). Intronic or synonymous variants outside of canonical splice sites (hereinafter referred to as noncanonical variants) with uncertain significance were identified in MYO5B posing a challenge in clinical interpretation. This study is aimed at assessing the effects of these variants on premessenger RNA (pre-mRNA) splicing to improve recognition of pathogenic spliceogenic variants in MYO5B and better characterize the MYO5B genetic variation spectrum. Disease-associated MYO5B noncanonical variants were collected from the literature or newly identified low GGT cholestasis patients. In silico splicing predictions were performed to prioritize potential pathogenic variants. Minigene splicing assays were performed to determine their splicing patterns, with confirmation by blood RNA analysis in one case. Eleven (five novel) noncanonical variants with uncertain significance were identified. Minigene splicing assays revealed that three variants (c.2090+3A>T, c.2414+5G>T, and c.613-11G>A) caused complete aberrations, five variants (c.2349A>G/p.(=), c.4221G>A/p.(=), c.1322+5G>A, c.1669-35A>C, and c.3045+3A>T) caused predominant aberrations, and three variants (c.4852+11A>G, c.455+8T>C, and c.2415-6C>G) had no effect on pre-mRNA splicing. Patient-derived RNA analysis showed consistent results. Based on our results, eight variants were reclassified as likely pathogenic and three as likely benign. Combining the clinical features and the above analysis, the diagnosis of MYO5B-associated FIC could be made in three new patients. In conclusion, we characterized the splicing patterns of MYO5B noncanonical variants and suggest that RNA analysis should be routinely included in clinical diagnostics to provide essential evidence for the interpretation of variants.