Background: Recent studies have suggested that ovarian stimulation and assisted reproductive techniques (ART) may increase the frequency of monozygotic twins. In this article, we present the analysis of the estimated frequency of twin deliveries following in vitro fertilization (IVF) in Lombardy during the period 2010–2014 for a total of 450,949 pregnancies. Method: This is a population-based study using data from the regional data base of Lombardy, a northern Italian region with a population of about 10 million inhabitants. During the considered period, a total of 461,424 single or multiple births were registered in Lombardy. After exclusion of triplets or more pregnancies, the total number of twin deliveries, in separate strata of like and unlike sex pregnancies twin deliveries, were obtained and the rate of twin deliveries was computed according to spontaneous and non-spontaneous conception and type of ART. Further, estimates of dizygotic or monozygotic twin births were calculated using Weinberg's methods. Results: The frequency of twins deliveries was 1.24/100 deliveries after natural conception and 20.05 after assisted conception. The estimated rates of monozygotic twins was 0.45 and 0.72/100 (95% CI: 0.58–0.91) deliveries after natural and assisted conception, respectively. This difference was statistically significant (p < .05). Conclusion: the present population based study suggests that the risk of monozygotic twin is about 60% higher among assisted than after natural conception.