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The milk containing only A2 β-casein (called A2 milk) is globally popular because of its unique health benefits. Traditionally, genetic testing (such as gene sequencing) is used to identify the cows with A2 β-casein gene that can only produce A2 milk, which is a time-consuming and costly method. The objective of this study was to directly identify A1 and A2 milk from a large quantity of milk using mid-infrared (MIR) spectroscopy and chemometrics without genotyping cows. Before establishing the predictive model, we firstly genotyped the A1 β-casein and A2 β-casein of cows from blood as reference values. Further, the MIR spectra of the milk collected from these cows were obtained using a dairy product analyzer. The MIR spectroscopy data and the reference values were used as the independent and dependent variables, respectively, to establish a category classification model for A1 and A2 milk. Seven preprocessing methods were combined with two feature extraction algorithms to establish the model. Subsequently, partial least squares discriminant analysis (PLS-DA) and support vector machine (SVM) models were developed. The average accuracy of the test set of the two models were 94.9% and 94.4%, respectively, while the PLS-DA model exhibited better effect, and the accuracy of training set and test set reached 96.6% and 96.0%, respectively. We used a set of independent samples for the external validation of the PLS-DA model, and the prediction accuracy was 95.2%. Overall, the proposed prediction models based on MIR spectroscopy can be used for low-cost, rapid, and large-scale classification of A1 and A2 milk, which may be extremely beneficial in milk production industries. • A1 and A2 milk identification was feasible using MIR spectroscopy. • UVE and CARS algorithm can used to extract spectral characteristic variables. • PLS-DA model exhibited better effect than SVM model. [ABSTRACT FROM AUTHOR]