Ancient Roman populations are expected to exhibit considerable biological variation because of extensive trade networks and migration patterns throughout Europe and the circum‐Mediterranean. The purpose of this research is to examine regional biological variation in Italy during imperial Roman times (1st to 3rd centuries ad) using three samples exhibiting distinctive class and economic systems. The individuals buried at Isola Sacra and Velia represent middle‐class tradesmen and merchants from coastal port populations from Central and Southern Italy, respectively, while the individuals from Castel Malnome represent an inland population near Rome of freed slaves and other lower‐class individuals involved in heavy labour associated with salt production. Data were recorded from 25 three‐dimensional cranial coordinate landmarks and analysed using Procrustes superimposition and associated multivariate statistics in MorphoJ. Procrustes analysis of variance statistics were unable to detect any significant group differences for centroid size (p = 0.595), but did detect differences in shape (p = 0.0154), suggesting some variation between the three samples. Canonical variates analyses based on Procrustes distance values suitable for small sample sizes indicated that while Castel Malnome was not significantly different from either of the coastal sites (Isola Sacra, p = 0.2071 and Velia, p = 0.8015), Isola Sacra and Velia were significantly different (p = 0.0119). The similarity of Castel Malnome to the other sites may reflect inherent heterogeneity in the sample, as it represents a group of freed slaves likely originating from various geographic locations. The separation of the two coastal sites is not unexpected, as Velia's location was influenced by an influx of Greek populations in the southern Italian Peninsula. This work supports previous studies using traditional craniometrics and dental metrics showing group similarity across the Italian Peninsula and a separation between more northern and southern groups. Copyright © 2017 John Wiley & Sons, Ltd.