Abstract Background While their incidence is on the rise, twin pregnancies are associated with risks to the mothers and their babies. This study aims to investigate the likelihood of adverse neonatal outcomes of twins following assisted reproductive technology (ART) compared to non-ART twins. Methods A retrospective population study using the Australian National Perinatal Data Collections (NPDC) which included 19,662 twins of ≥20 weeks gestational age or ≥ 400 g birthweight in Australia. Maternal outcomes and neonatal outcomes (preterm birth, low birth weight, resuscitation and neonatal death) were compared. Generalized Estimating Equations were used to assess the likelihood of any neonatal outcomes, with adjusted odds ratio (AOR) and 95% confidence intervals (CI) presented. Weinberg’s differential rule was used to estimate monozygotic twin rate. Results ART mothers were 3.3 years older than non-ART mothers. The rates of pregnancy-induced hypertension and gestational diabetes were significantly higher for ART mothers than non-ART mothers (12.2% vs. 8.4%, p