Here, we report results on systematic analysis of DNA substrate preferences of three N6-adenine β-class DNA methyltransferases that are part of the type II restriction-modification systems. The studied enzymes were: M.EcoVIII, M.HindIII and M.LlaCI, which although found in phylogenetically distant bacteria (γ-proteobacteria and low-GC Gram-positive bacteria), recognize the same palindromic specific sequence 5'-AAGCTT-3' and catalyze formation of N6-methyladenine at the first A-residue. As expected overall the enzymes share the most analyzed features, but they show also some distinct differences in substrate recognition. Therefore DNA methylation reactions were carried out not only under standard, but also under relaxed conditions using DMSO or glycerol. We found that all of these enzymes preferred DNA containing a hemimethylated target site, but differ in modification of ssDNA, especially more pronounced for M.EcoVIII under relaxed conditions. In these conditions they also have shown varied preferences toward secondary sites, which differ by one nucleotide from specific sequence. They preferred sequences with substitutions at the 1st (A1 → G/C) and at the 2nd position (A2 → C), while sites with substitutions at the 3rd position (G3 → A/C) were modified less efficiently. Kinetic parameters of the methylation reaction carried out by M.EcoVIII were determined. Methylation efficiency (kcat/Km) of secondary sites was 4.5-10 times lower when compared to the unmethylated specific sequences, whilst efficiency observed for the hemimethylated substrate was almost 4.5 times greater. We also observed a distinct effect of analyzed enzymes on unspecific interaction with DNA phosphate backbone. We concluded that for all three enzymes the most critical is the phosphodiester bond between G3-C4 nucleotides at the center of the target site.