Low pathogenic avian influenza viruses (LPAIVs) are genetically highly variable and have diversified into multiple evolutionary lineages that are primarily associated with wild bird reservoirs. Antigenic variation has been described for mammalian influenza viruses and for highly pathogenic avian influenza viruses that circulate in poultry, but much less is known about antigenic variation of LPAIVs. In this study, we focussed on H13 and H16 LPAIVs that circulate globally in gulls. We investigated the evolutionary history and intercontinental gene flow based on the hemagglutinin (HA) gene and used representative viruses from genetically distinct lineages to determine their antigenic properties by hemagglutination inhibition assays. For H13 at least three distinct genetic clades were evident, while for H16 at least two distinct genetic clades were evident. Twenty and ten events of intercontinental gene flow were identified for H13 and for H16 viruses, respectively. At least two antigenic variants of H13 and at least one antigenic variant of H16 were identified. Amino acid positions in the HA protein that may be involved in the antigenic variation were inferred, and some of the positions were located near the receptor binding site of the HA protein, as they are in the HA protein of mammalian influenza A viruses. These findings suggest independent circulation of H13 and H16 subtypes in gull populations as antigenic patterns do not overlap and contribute to the understanding of the genetic and antigenic variation of LPAIV naturally circulating in wild birds.IMPORTANCE Wild birds play a major role in the epidemiology of low pathogenic avian influenza viruses (LPAIVs) from which these viruses are occasionally transmitted-directly or indirectly-to other species, including domestic animals, wild mammals and humans, where they can cause subclinical to fatal disease. Despite a multitude of genetic studies, the antigenic variation of LPAIVs in wild birds is poorly understood. Here, we investigated the evolutionary history, intercontinental gene flow, and the antigenic variation among H13 and H16 LPAIVs. The circulation of the subtypes H13 and H16 seems to be maintained by a narrower host range, in particular gulls, than for the majority of LPAIV subtypes and may therefore serve as a model for evolution and epidemiology of H1-H12 LPAIVs in wild birds. The findings suggest that H13 and H16 LPAIVs circulate independently of each other and emphasize the need to investigate within clade antigenic variation of LPAIVs in wild birds.