Gene clustering based on amino acid sequence similarity has been one of the most important problems and always challenging in molecular biology. The most conventional methods are based on alignment-technique. These methods cannot identify and classify sequences, especially when the lengths of sequence are long and unequal. Therefore, in order to classify fungal hexosaminidase amino acid sequences and put them in the right taxonomical group we evaluate the feasibility of computational free alignment methods based on machine learning classifiers such as SVM, KNN, SOM and ensemble technique. The classifiers have appropriately categorized large Dikarya hexosaminidase amino acid sequences as data sets according to their taxonomical groups in two phyla named, the “Ascomycota” and the “Basidiomycota”. Two statistical methods including paired t-test and PCA were used for the feature selection and reduce the dimensionality of the features, respectively. Seven classifier performance metrics, randomized complete block design (RCBD), pairwise Tukey’s honestly significant difference (HSD) tests and the technique for order preference by similarity to ideal solution (TOPSIS) with modified k-fold cross validation have been used as tools in order to evaluate and ranking of classifiers. In this study, the effect of training data size on the classifier performance was investigated. The results showed that the rank and the performance of classifiers were depended on the training data size. The highest obtained values for the average overall accuracy of the following training data sizes, 80%, 60%, 40% and 20% using KNN, KNN, ensemble and ensemble classifier were 96.96%, 95.81%, 94.47% and 92.47%, respectively.

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