<p>Traditional breeding, encompassing sexual hybridization and subsequent selection of favorable traits in plants, has predominantly served as the primary approach for <i>Vanilla</i> breeding. However, alternative methods, including mutation induction via physical and chemical mutagens under in vitro conditions, as well as biotechnological techniques like somaclonal variation and genetic transformation, have also been explored and implemented in <i>Vanilla</i> breeding initiatives. This review aims to study the current technique and applications of in vitro mutagenesis in <i>Vanilla</i>, including the progress and influencing factors of in vitro mutagenesis technique, as well as advances, challenges and prospects of mutagenesis breeding under in vitro conditions. In this regard, plant tissue culture techniques have been an innovative tool to overcome these problems. Effective protocols for in vitro propagation or micropropagation, in vitro conservation, cryopreservation, somaclonal variations, protoplast culture, in vitro mutagenesis, genetic transformation, and genome editing of <i>vanilla</i> species are available. The analysis reveals that targeted mutagenesis approaches, particularly gene-editing tools, offer the most significant potential for precise trait enhancement in <i>vanilla</i>. These findings are important to develop new hybrids with improved traits. </p>