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Patterns of seasonal variation in births in some neuropsychiatric conditions have been found in previous research; however, no study to date has examined these disorders for seasonal variation in singletons and multiple births separately. This study aimed to determine whether the birth date distribution for individuals with autism spectrum disorders (ASD), including singletons and multiple births, differed from the general population. Two ASD case groups were studied: 907 singletons and 161 multiple births concordant for ASD. Two control groups were obtained from registered births of singletons and multiples. Results of the non-parametric time-series analyses, where day of birth was used, suggested there were three peaks in ASD singletons and ASD concordant multiple births. Roughly, the peaks were in April, June and October for singletons and about 2-4 weeks earlier in multiples. Results from multivariable Poisson regression, where month of birth was used, indicated that ASD concordant multiple births in males tended to be higher than expected in March, May and September (with borderline statistical significance), but were 87% less in December (P < 0.05), as compared with January. Overall, the patterns of relative risk estimates from Poisson regression are similar to findings from the non-parametric time-series approach, but are not exactly congruent. It is important to note that indications of seasonality may be sensitive to the selection of time cut-points and therefore an arbitrary binning of time can either mask existing trends or falsely indicate the presence of a trend. The presence of seasonal trends in ASD singletons and concordant multiple births suggests a role for non-heritable factors operating during the pre- or perinatal period, even among cases with a genetic susceptibility.