Plant off-types still become a concern in pineapple micropropagation. Reliable methods are needed to detect and to reduce plant off-types. This research was conducted to confirm the occurrence of somaclonal variation during micropropagation, to know the level of variation in three different populations, to detect and to reduce the somaclonal variation of pineapple clone Simadu. The first population was the acclimatized plants and plantlets of four-year old cultures. The second population was the plantlets yielded from three kinds of regeneration methods (direct organogenesis, indirect organogenesis, somatic embryogenesis) derived from the normal phenotype shoots selected from the first population. The third population was the new in vitro cultures regenerated by shoot proliferation and direct organogenesis methods. RAPD analysis by using 10 primers was performed to confirm genetic variation. The result showed that the phenotypic variation occurred in the four year-old pineapple plants and plantlets were due to genetic variation. This research has proved that the long-period of cultures is the main contributor of somaclonal variation, while the regeneration method and plant growth regulator could also induce genetic variation. The new cultures showed higher level of similarity. Therefore, it needs a correct strategy to apply micropropagation method.