•Effects of nucleotide sites in acid damage of DNA were studied with MALDI MS.•Terminal nucleotides were easily damaged than middle ones in MALDI MS.•5′-Terminal was easily damaged than 3′-ones in the further step in MALDI MS.Acid hydrolysis of DNA oligonucleotides leads to nucleotides damage, especially affecting those containing purine bases. Previous research focused mainly on the types of cleaved nucleobases and the cleavage efficiency. There has been no report about the nucleotide site. In this study, 10 DNA oligonucleotides with differing dAMP and dGMP sites were synthesized to study the influence of these sites with MALDI-TOF MS, which offers a powerful tool with advantages of rapid speed, high sensitivity, high tolerance and simple mass spectra. Results show that, with acid hydrolysis, cleavage of terminal nucleotides differs from those in the middle in the MALDI-TOF mass spectra. In addition, in the course of further degradation, there are obvious differences between nucleotides at the 5′ and 3′-termini.