USDA confirmed by whole genome sequence the first detection of HPAI H5N1 clade 2.3.4.4b genotype D1.1 in dairy cattle. While genotype D1.1 has been the dominant strain circulating in migratory birds in North America, the Nevada cases represent the first detection of a genotype other than B3.13 in cattle and the second known spillover event from wild birds into lactating dairy cattle. D1.1 clinical presentation in dairy herds in both Nevada and Arizona was mild compared to HPAI B3.13. However, this is based on a small number of affected herds and may not be the case for the broader population. Here we sought to experimentally reproduce infection of dairy cattle with HPAI H5N1 genotype D1.1. and also sought determine if cattle with serum antibodies following natural infection with HPAI B3.13 were protected against reinfection with HPAI D1.1. Four adult Holstein lactating cows were moved into ABSL-3-Ag containment, two cows free of influenza A virus and two cows free of influenza A virus, but with serum antibodies from a natural H5N1 infection (genotype B3.13 ). All cows were inoculated via the intramammary route with 1 ml of 1 x 105.4 TCID50/ml A/dairy cattle/Nevada/24-002644-003/2025 into two contralateral quarters. The drop in milk production and rumination observed in this study were similar to those reported in experimental intramammary challenge of lactating cows with HPAI B3.13, as well as natural infections, indicating that clinical presentation of HPAI D1.1 was similar in severity to experimental challenge with HPAI B3.13. Unlike the HPAI B3.13 intramammary challenges, HPAI D1.1 migrated and infected a non-inoculated quarter. The two B3.13 convalescent cows were susceptible to reinfection with D1.1, demonstrating clinical signs including a drop in milk production and rumination, pyrexia, and mastitis. However, milk production and rumen motility recovered more quickly in the two convalescent cows than in the two na?ve cows and pyrexia was not as severe. Viral RNA was also not detected in the milk of the convalescent cows after 10 DPI while it was detected in the milk of the na?ve cows for the durations of the study. Furthermore, while viral RNA was detected in the milk of both convalescent cows, no viable virus was isolated. While convalescent cows with serum but not milk antibodies to B3.13 are susceptible to reinfection with D1.1 and clinical disease antibodies can transudate into the milk and bind virus, likely preventing further spread throughout the herd. The single-nucleotide variant analyses of whole genome sequences virus recovered from the milk of previously na?ve cows also uncovered some potentially important patterns. Genes HA and MP were found to have strong evidence for natural selection and analysis indicates a fitness advantage is conferred through some key mutations that could lead to antigenic drift and immune escape.