Atomic force microscopy (AFM) has many advantages in the study of biological samples, such as the convenient specimen preparation and the high resolution. In the present study, AFM was used to observe the double minute chromosomes (DMs) in mouse methotrexate-resistant cell line 3T3R500. AFM images were obtained by tapping mode, contact mode and later force mode of AFM. DMs were composed of two compact spheres connected with fibers. The number of DMs in the 3T3R500 cells increased with increasing levels of methotrexate (MTX) resistance. The data of the height and the underside diameter of the DMs were also obtained. The details of specimen preparation and scan mode selection of AFM were discussed. Our results show that AFM is a powerful method in the study of DMs.