The origins and relationships of Newcastle disease virus (NDV) vaccine strains Hertfordshire (H) and Mukteswar, and the virulent Herts'33 were studied using partial sequence analysis of the fusion protein gene. The mesogenic strain H was obtained by egg passages of a field virus isolated in England in 1933 (later known as Herts'33). Different lines of the strain Herts'33, however, divided into two distinct groups: genotype IV, and a hitherto undescribed lineage, which comprised the Weybridge line (Herts'33/56). Vaccine strain H and the two clusters comprising viruses designated Herts'33 displayed 6.5 to 6.8% and 15.6 to 16.3% mutational distances, respectively, which precluded parent-offspring relationships with either of them. In contrast, the different lines of the vaccine strain Mukteswar, which was reportedly derived from an Indian field isolate in the mid-1940s, showed 98.9 to 100% sequence similarity to strain H. It is therefore probable that the two vaccines were derived from the same virus stock.