De novo marker chromosomes and/or subtle structural aberrations may be recognized but not defined by routine banding or FISH analysis, and this creates difficulties in specific diagnosis and in pediatric genetic counseling. In this study, we applied a direct and efficient approach, micro-FISH, in identifying the origin and composition of three de novo chromosomal aberrations. Five to 10 copies of the aberrant chromosome were collected by microdissection with the DNA amplified by PCR and labeled with biotin/digoxiginin, and then hybridized to normal metaphase spreads. A de novo marker chromosome was identified as inv dup (15) (pter → q11::q11→pter) in a 2 year old child with seizure disorders, agenesis of corpus collosum and developmental delay. A der (9)t(9;9)(pter→q21.2::p13→pter) was found in a 13 year old child with shunted hydrocephalus, deep palmer creases, marbled pigmentation, and developmental delay. Additionally, an interstitial deletion of 6q16.2q21 was found in the blood specimen of a 2 year old with mental and psychomotor delay and obesity. Our study reveals that micro-FISH can be practically applied in pediatric genetic diagnosis by identifying the de novo aberrations that could not be defined by routine cytogenetic analysis.