Concerns have been raised about a potential association between ARTs and hypospadias. Since same sex twins share the same in utero exposures to medications and maternal factors, they provide an opportunity to discover whether environmental or genetic factors play the dominant role in the pathogenesis of this congenital anomaly. Although the spontaneous twinning rate in the general population is 1.25%; 30% of live births following ART are twins. This becomes a public health matter since twin pregnancies have twice the congenital malformation rate of singletons. The most recent NY state report has 4% incidence of neonatal malformations among all live births and 8% in twin pregnancies. We have examined whether differences in hypospadias grade severity exist between singletons and twins, and whether ART influences the incidence of hypospadias in second male twins or the severity of hypospadias in affected twins from 07/1996 to 03/ 2006 at a university based Pediatric Urology Center. Patient data included medical and surgical histories such as congenital anomalies, gestational order, family history, parental medical, fertility and obstetric history were reviewed. Patients were first separated into singleton and twin groups. The twin group was further subdivided into opposite- or same-gender twins. The singleton and same gender twin groups were classified according to the conception method: ART or naturally conceived. Finally, these groups were categorized by hypospadias grade as mild (glandular or coronal), or severe (midshaft, penoscrotal or perineal). Comparisons between singletons and twins revealed no differences in hypospadias grade severity, whether analyzing only ART singletons and twins or naturally conceived singletons and twins. The database included 227 patients, 193 (85%) singletons and 34 (15%) twins comprising 30 twin sets. Of the 30 twin sets, 18 (60.0%) were of the same gender, and 12 (40.0%) were opposite gender twin sets. We further examined only the same gender twin sets. All of the 9 (100%) sets conceived by ART were dizygous and discordant for hypospadias; only one twin being affected, whereas among the naturally conceived twins, 4 (44.4%) were monozygous and 5 (55.6%) were dizygous. In three of the four monozygous twin sets, both twins were affected with hypospadias and in 4 (80%) of the dizygous twin sets, only one twin was affected. Although none of the considered aggressors proved to have any role in the occurrence and severity of hypospadias, a tendency toward being discordant in dizygous gestations and concordant in monozygous twins was confirmed. In assessing the severity of the hypospadias, the twins needed to be evaluated individually because even if they were concordant for the occurrence of hypospadias, they were discordant for grade. There was no difference in the severity of the anomaly between the ART and naturally conceived same gender twins. The occurrence of twins does not appear to increase the severity of hypospadias, nor does the conception method (natural or ART) correlate with the risk of hypospadias in second male twins or its type in those affected. Interestingly, the pattern the discordant presence of hypospadias in dizygotic twin pregnancies may absolve ART of any responsibility in the increased incidence of penile anomalies, while the consistent appearance of the anomaly in both monozygotic twins, appears to suggest a genetic origin.