For a study, researchers sought to understand that Rett syndrome (RTT) is a neurodevelopmental disorder most commonly caused by a pathogenic variant in the X-linked MECP2 gene. Internalizing behaviors appear to be common, but standard methods of diagnosing anxiety were difficult to apply in the population, with cognitive impairment and limited expressive language. The study aimed to describe the frequency of anxiety-like behavior and anxiolytic treatments and associated clinical features in people with RTT. From 2006 to 2019, 1,380 females with RTT participated in 2 iterations of the multicenter United States RTT Natural History Study (RNHS) using parental reports and medication logs. The majority of RTT participants (77.5%) displayed at least occasional anxious or nervous behavior. Anxiety was reported as the most troublesome concern for 2.6% of participants in the second iteration and 1 of the top 3 concerns for 10.0%. In the second iteration, 16.6% of participants’ parents directly reported treatment for anxious or nervous behavior, with the majority reporting good control of the behavior (71.6%). Anxiety was listed in the medication logs of a similar number of participants in both RNHS iterations (15% and 14.5%, respectively). Anxiolytic and selective serotonin reuptake inhibitor (SSRI) use was associated with more anxiety-like behaviors (P<0.001), older age (P<0.001), and mild MECP2 variants (P=0.002). Anxiety-like behavior was common at all ages and was a major source of parental concern in RTT. Medication was more likely to be used in older people and those with mild MECP2 variants. Better anxiety diagnosis and treatment in RTT should be a goal of future research and clinical care.