Benzene is a well-known carcinogen that induces chromosomal instability, including chromosome aberration and aneuploidy. In order to assess aneugenic effect of low-level benzene, micronucleus-centromere assay using specific probes for chromosomes 7 and 9 was performed in workers occupationally exposed to low-dose benzene at a petroleum refinery. A micronucleus-centromere assay using a pan-centromeric probe was also performed to determine the origin of benzene-induced micronucleus (MN). Frequency of aneuploidy of chromosomes 7 and 9 was significantly higher among workers compared to the unexposed control group. Poisson regression analysis revealed that aneuploidy frequency of chromosome 7 or 9 was significantly associated with benzene level after adjusting for confounding variables such as age, smoking, alcohol intake, and duration of work (p = .042). Additionally, frequencies of MN and centromere-negative micronuclei (MNC-) were significantly higher in benzene-exposed workers compared to controls, while frequency of centromere-positive micronuclei (MNC+) was similar in both groups. In conclusion, aneuploidy of chromosomes 7 and 9 could be a useful biomarker to assess the effect of low-level benzene exposure, and benzene-induced MN originates from chromosome breaks rather than chromosome non-disjunction.