Some studies have confirmed the variation in milk profiles when dairy cows show estrus. However, only a few milk components, such as fat, protein, and lactose, have been investigated so far, and thus any changes in the many other parts of milk’s composition due to estrus are unknown. Milk mid-infrared (MIR) spectra consist of wavenumbers, which provide insight into the chemical composition of milk. The MIR spectrum reflects the global composition of milk, but this information is currently underused. In this study, we considered MIR wavenumbers as traits, and directly studied the spectral information as a way to study the estrus of dairy cows linked to milk composition. This research provides a deeper understanding of the milk MIR spectrum and may lead to new approaches for estrus detection in dairy cows from routine milk analysis, thereby guiding an opportune insemination time