D. subobscura, a typically Palearctic species, has recently colonized the American continent. The population from which the colonization started is still unknown. A complete association between a lethal gene and the O5 chromosomal inversion has been detected in the colonizing populations; this association could be used to ascertain the origin of the colonization. In the present study, three hypotheses on the origin of this association are analyzed and their probabilities are calculated. According to the first hypothesis an O5 chromosome carrying a lethal gene would have been included in the sample from which the colonization started, thus originating the association found in the American populations. The second hypothesis assumes that, at the beginning of the colonization, the association between the O5 inversion and a lethal gene would have been formed from an Ost chromosome carrying a lethal gene, on which a new O5 (or similar) inversion arose. In the last hypothesis, the association between the O5 inversion and the lethal gene would have originated at the beginning of the colonization from a lethal-free O5 chromosome, in which a lethal gene arose due to mutation.