Abstract: In the present study, in order to explore the possibility of distant hybridization of Micropterus salmoides, artificial insemination was used and hybrid F₁ was successfully obtained from distant hybridization between female largemouth bass and male Lepomis mearchirus. The hybrid F₁ embryos underwent apparently normal morphological development. The hatching time of hybrid F₁ was about 49 h when the water temperature was (22.5±0.5) °C. The fertilization rate was 65% and the hatching rate was 21%. In hybrid F₁, the average weight gain was 1.55 g/d, and the fodder utilization rate was 1.24 after a ten-week in-door cultivation. The hybrid F₁ showed a similar growth rate with maternal parent M. salmoides but statistically significantly higher than paternal L. mearchirus. The morphological characteristics of hybrid F₁ showed obvious hybridization properties. The number traits (numbers of lateral line scales, dorsal fins, pectoral fins and anal fins) and metric traits (measurable traits and frame metric traits) in hybrid F₁ showed an intermediate level between the two parents. A number of metric traits biased towards paternal L. mearchirus more than maternal M. salmoides was observed in hybrid F₁. In addition, several morphology traits of hybrid F₁ exceeded both of the two parents. 5S rDNA gene amplification and analysis showed that the two parents possess two different types of 5S rDNA, respectively, while the hybrid F₁ inherits four types of 5S rDNA from parents. The four types of 5S rDNA coding region sequences (CDS, 120 bp) are highly conserved. Two types of 5S rDNA transcribed spacer (NTS) from hybrid F₁ and maternal parent M. salmoides had a base "GTC" variable region (sequence length ranged from 186 to 205 bp), while two types of 5S rDNA NTS from hybrid F₁ and paternal parent L. mearchirus are highly conserved (sequence length was 86 and 263 bp, respectively). These 5S rDNA types confirmed that the hybrid F₁ fused genomes of the two parents. Our study provides a new perspective in exploring distant hybridization of M. salmoides.