Tomato (Solanum lycopersicum L.) is a major horticultural crop and a model species among eudicots, especially for traits related to reproductive development. Although considerable progress has been made since the tomato genome sequence project was completed, most of the genes identified remain predictions with an unknown or hypothetical function. This lack of functional characterization hampers the use of the huge amount of genomic information available to improve the quality and productivity of this crop. Reverse genetics strategies such as artificial mutagenesis and next-generation sequencing approaches build the perfect tandem for increasing knowledge on functional annotation of tomato genes. This work reports the phenotypic characterization of a tomato mutant collection generated from an EMS chemical mutagenesis program aimed to identify interesting agronomic mutants and novel gene functions. Tomato mutants were grouped into fourteen phenotypic classes, including vegetative and reproductive development traits, and the inheritance pattern of the identified mutations was studied. In addition, causal mutation of a selected mutant line was isolated through a mapping-by-sequencing approach as a proof of concept of this strategy’s successful implementation. Results support tomato mutagenesis as an essential tool for functional genomics in this fleshy-fruited model species and a highly valuable resource for future breeding programs of this crop species aimed at the development of more productive and resilient new varieties under challenging climatic and production scenarios.